Uses and compositions for treatment of ankylosing spondylitis

ABSTRACT

The invention provides methods, uses and compositions for the treatment of ankylosing spondylitis (AS). The invention describes methods and uses for treating ankylosing spondylitis, wherein a TNFα inhibitor, such as a human TNFα antibody, or antigen-binding portion thereof, is used to reduce signs and symptoms of ankylosing spondylitis in a subject. Also described are methods for determining the efficacy of a TNFα inhibitor for treatment of ankylosing spondylitis in a subject.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 60/812,312, filed on Jun. 8, 2006; U.S. Provisional PatentApplication No. 60/857,352, filed on Nov. 6, 2006; and U.S. ProvisionalPatent Application No. 60/858,328, filed on Nov. 10, 2006.

BACKGROUND OF THE INVENTION

Ankylosing spondylitis (AS) is a chronic, progressive, inflammatorydisease with considerable impact on patient functioning, well-being, anddisability. The prevalence of AS has traditionally been estimated in therange of 0.1-1.9%, with more males affected than females (Sieper et al.Ann Rheum Dis 2001; 60:3-18; Silmani & Hochberg Rheum Dis Clin North Am1996; 22:737-49; Gran & Husby, Semin Arthritis Rheum 1993;22(5):319-34). Millions of people are affected by ankylosing spondylitis(AS). As a chronic disease of the axial skeleton and large peripheraljoints, AS causes inflammatory back pain and stiffness and it isassociated with other inflammatory diseases of the skin, eyes andintestines. AS is difficult to diagnose in its early stages and is oftenan overlooked cause of persistent back pain in young adults. In severecases, AS may result in complete spinal fusion, causing extreme physicallimitation. Thus, there remains a need for a safe and effectivetreatment for AS.

As the disease progresses, patients with AS experience pain, jointstiffness, and the eventual loss of spinal mobility. These clinicalsymptoms and subsequent disease progression result in functionallimitations and impairment in health-related quality of life (HRQOL)(Dagfinrud et al. Ann Rheum Dis 2004:63:1605-10; Bostan et al. RheumatolInt 2003; 23:121-6; Zink et al., J Rheumatol 2000; 27:613-22; Ward 1998,Rheum Dis Clin North Am 1998; 24:815-27) and work productivity (Boonenet al. Ann Rheum Dis 2002; 61:429-37; Boonen et al. J Rheumatol 2001;28:1056-62).

No cure exists for AS. Generally, treatment includes trying to relievepain and stiffness using medications such as nonsteroidalanti-inflammatory drugs (NSAIDs), corticosteroids, and disease-modifyingantirheumatic drugs (DMARDs). In recent years biologic responsemodifiers that inhibit TNF activity have become established therapiesfor AS.

SUMMARY OF THE INVENTION

Although TNFα inhibitors are effective at treating AS, there remains aneed for a more effective treatment option for subjects suffering fromAS, especially in improving the fatigue and pain associated with thedisease and in treating subjects who have failed more conventional,i.e., DMARD or NSAIDs therapy. Thus, there also remains a need forimproved methods and compositions that provide a safe and effectivetreatment of AS using TNFα inhibitors.

The instant invention provides improved methods and compositions fortreating AS. The invention further provides a means for treating certainsubpopulations of patients who have AS. The invention further provides ameans by which the efficacy of a TNFα inhibitor for the treatment of AScan be determined. Each of the examples described herein describesmethods and compositions which can be used to determine whether a TNFαinhibitor is effective for treating the given disorder, i.e. AS.

The invention provides a method of decreasing pain and fatigue in asubject having AS comprising administering a human TNFα antibody, orantigen-binding portion thereof, to the subject such that pain andfatigue are decreased. In one embodiment, the decrease in fatigue in thesubject is determined by a score selected from the group consisting ofFACIT-F, BASDAI, and SF-36. In one embodiment, the decrease in fatigueis determined by a decrease of at least about 1.9 in a BASDAI score ofthe subject. In one embodiment, the decrease in fatigue is determined bya decrease of at least about 2.0 in a BASDAI score of the subject.

The invention also provides a method of inducing partial remission of ASin a subject comprising administering a human TNFα antibody, orantigen-binding portion thereof, to the subject, such that partialremission of AS is induced.

The invention further provides a method of treating AS in a subject whohas failed either DMARD therapy or NSAIDs therapy comprisingadministering a human TNFα antibody, or antigen-binding portion thereof,to the subject, such that AS is treated.

The invention includes methods for determining the efficacy of a TNFαinhibitor for treating ankylosing spondylitis (AS) in a subjectcomprising determining a partial remission rate of a patient populationhaving AS and who was administered the TNFα inhibitor, wherein a partialremission rate of at least about 20% of the patient population indicatesthat the TNFα inhibitor is an effective TNFα inhibitor for the treatmentof AS.

The invention further provides a method of determining the efficacy of aTNFα inhibitor for treating ankylosing spondylitis (AS) in a subjectcomprising determining a Bath Ankylosing Spondylitis Disease ActivityIndex (BASDAI) 20 response of a patient population having AS and who wasadministered the TNFα inhibitor, wherein a BASDAI 20 response in atleast about 60% of the patient population indicates that the TNFαinhibitor is an effective TNFα inhibitor for the treatment of AS. In oneembodiment, a BASDAI 20 response in at least about 70% of the patientpopulation indicates that the TNFα inhibitor is an effective TNFαinhibitor for the treatment of AS. In one embodiment, a BASDAI 20response in at least about 80% of the patient population indicates thatthe TNFα inhibitor is an effective TNFα inhibitor for the treatment ofAS. In one embodiment, a BASDAI 20 response in at least about 85% of thepatient population indicates that the TNFα inhibitor is an effectiveTNFα inhibitor for the treatment of AS.

The invention includes a method of determining the efficacy of a TNFαinhibitor for treating ankylosing spondylitis (AS) in a subjectcomprising determining a Bath Ankylosing Spondylitis Disease ActivityIndex (BASDAI) 50 response of a patient population having AS and who wasadministered the TNFα inhibitor, wherein a BASDAI 50 response in atleast about 23% of the patient population indicates that the TNFαinhibitor is an effective TNFα inhibitor for the treatment of AS. In oneembodiment, a BASDAI 50 response in at least about 30% of the patientpopulation indicates that the TNFα inhibitor is an effective TNFαinhibitor for the treatment of AS. In one embodiment, a BASDAI 50response in at least about 40% of the patient population indicates thatthe TNFα inhibitor is an effective TNFα inhibitor for the treatment ofAS. In one embodiment, a BASDAI 50 response in at least about 50% of thepatient population indicates that the TNFα inhibitor is an effectiveTNFα inhibitor for the treatment of AS. In one embodiment, a BASDAI 50response in at least about 60% of the patient population indicates thatthe TNFα inhibitor is an effective TNFα inhibitor for the treatment ofAS.

The invention further provides a method of determining the efficacy of aTNFα inhibitor for treating ankylosing spondylitis (AS) in a subjectcomprising determining a Bath Ankylosing Spondylitis Disease ActivityIndex (BASDAI) 70 response of a patient population having AS and who wasadministered the TNFα inhibitor, wherein a BASDAI 70 response in atleast about 10% of the patient population indicates that the TNFαinhibitor is an effective TNFα inhibitor for the treatment of AS. Theinvention also includes a BASDAI 70 response in at least about 20% ofthe patient population indicates that the TNFα inhibitor is an effectiveTNFα inhibitor for the treatment of AS. In one embodiment, a BASDAI 70response in at least about 30% of the patient population indicates thatthe TNFα inhibitor is an effective TNFα inhibitor for the treatment ofAS. In one embodiment, a BASDAI 70 response in at least about 40% of thepatient population indicates that the TNFα inhibitor is an effectiveTNFα inhibitor for the treatment of AS. In one embodiment, a BASDAI 70response in at least about 45% of the patient population indicates thatthe TNFα inhibitor is an effective TNFα inhibitor for the treatment ofAS.

The invention describes a method of determining the efficacy of a TNFαinhibitor for treating ankylosing spondylitis (AS) in a subjectcomprising determining a Assessment in Ankylosing Spondylitis (ASAS) 20response of a patient population having AS and who was administered theTNFα inhibitor, wherein an ASAS20 response in at least about 27% of thepatient population indicates that the TNFα inhibitor is an effectiveTNFα inhibitor for the treatment of AS. In one embodiment, an ASAS20response in at least about 50% of the patient population indicates thatthe TNFα inhibitor is an effective TNFα inhibitor for the treatment ofAS. In one embodiment, an ASAS20 response in at least about 55% of thepatient population indicates that the TNFα inhibitor is an effectiveTNFα inhibitor for the treatment of AS. In one embodiment, an ASAS20response in at least about 70% of the patient population indicates thatthe TNFα inhibitor is an effective TNFα inhibitor for the treatment ofAS.

The invention provides a method of determining the efficacy of a TNFαinhibitor for treating ankylosing spondylitis (AS) in a subjectcomprising determining a Assessment in Ankylosing Spondylitis (ASAS) 40response of a patient population having AS and who was administered theTNFα inhibitor, wherein an ASAS40 response in at least about 10% of thepatient population indicates that the TNFα inhibitor is an effectiveTNFα inhibitor for the treatment of AS. In one embodiment, an ASAS40response in at least about 20% of the patient population indicates thatthe TNFα inhibitor is an effective TNFα inhibitor for the treatment ofAS.

In one embodiment, an ASAS40 response in at least about 30% of thepatient population indicates that the TNFα inhibitor is an effectiveTNFα inhibitor for the treatment of AS. In one embodiment, an ASAS40response in at least about 45% of the patient population indicates thatthe TNFα inhibitor is an effective TNFα inhibitor for the treatment ofAS in the subject.

The invention includes a method of determining the efficacy of a TNFαinhibitor for treating ankylosing spondylitis (AS) in a subjectcomprising determining a Assessment in Ankylosing Spondylitis (ASAS) 70response of a patient population having AS and who was administered theTNFα inhibitor, wherein an ASAS70 response in at least about 5% of thepatient population indicates that the TNFα inhibitor is an effectiveTNFα inhibitor for the treatment of AS. In one embodiment, an ASAS70response in at least about 20% of the patient population indicates thatthe TNFα inhibitor is an effective TNFα inhibitor for the treatment ofAS in the subject. In one embodiment, an ASAS70 response in at leastabout 23% of the patient population indicates that the TNFα inhibitor isan effective TNFα inhibitor for the treatment of AS in the subject. Inone embodiment, an ASAS70 response in at least about 30% of the patientpopulation indicates that the TNFα inhibitor is an effective TNFαinhibitor for the treatment of AS in the subject. In one embodiment, anASAS70 response in at least about 40% of the patient populationindicates that the TNFα inhibitor is an effective TNFα inhibitor for thetreatment of AS in the subject.

In one embodiment, the invention further comprises administering theeffective TNFα inhibitor to a subject to treat AS.

The invention also includes a method of treating AS in a subjectcomprising administering an effective TNFα inhibitor to the subject suchthat AS is treated, wherein the effective TNFα inhibitor was previouslyidentified as resulting in a BASDAI 20 response in at least about 60% ofa patient population having AS and who was administered the TNFαinhibitor.

The invention further provides a method of treating AS in a subjectcomprising administering an effective TNFα inhibitor to the subject suchthat AS is treated, wherein the effective TNFα inhibitor was previouslyidentified as resulting in a BASDAI 50 response in at least about 23% ofa patient population having AS and who was administered the TNFαinhibitor.

The invention provides a method of treating AS in a subject comprisingadministering an effective TNFα inhibitor to the subject such that AS istreated, wherein the effective TNFα inhibitor was previously identifiedas resulting in a BASDAI 70 response in at least about 10% of a patientpopulation having AS and who was administered the TNFα inhibitor.

The invention also provides a method of treating AS in a subjectcomprising administering an effective TNFα inhibitor to the subject suchthat AS is treated, wherein the effective TNFα inhibitor was previouslyidentified as resulting in an ASAS20 response in at least about 50% of apatient population having AS who was administered the TNFα inhibitor.

The invention includes a method of treating AS in a subject comprisingadministering an effective human TNFα antibody, or antigen-bindingportion thereof, to the subject, wherein the effective human TNFαantibody, or antigen-binding portion thereof, was previously identifiedas achieving an ASAS50 response in at least about 39% of a patientpopulation having AS who was administered the human TNFα antibody, orantigen-binding portion thereof.

The invention also includes a method of treating AS in a subjectcomprising administering an effective human TNFα antibody, orantigen-binding portion thereof, to the subject, wherein the effectivehuman TNFα antibody, or antigen-binding portion thereof, was previouslyidentified as achieving an ASAS70 response in at least about 5% of apatient population having AS who was administered the human TNFαantibody, or antigen-binding portion thereof.

The invention provides a method for monitoring the effectiveness of aTNFα inhibitor for the treatment of fatigue in a human subject having AScomprising administering the TNFα inhibitor to the subject; anddetermining the effectiveness of the TNFα inhibitor using a baselineFACIT-fatigue score and a FACIT-fatigue score following administrationof the TNFα inhibitor, wherein either a change of at least about 7 forthe FACIT-fatigue score indicates that the TNFα inhibitor is effectiveat reducing fatigue in a subject having AS.

The invention includes a method of testing the effectiveness of a TNFαinhibitor for decreasing fatigue in a patient having AS, comprisingcomparing a pre-determined FACIT-fatigue score following treatment ofthe patient with the TNFα inhibitor, with a pre-determined FACIT-fatiguebaseline score, wherein a change of at least about 7 indicates the TNFαinhibitor is effective for decreasing fatigue in a patient having AS.

The invention includes a method of achieving partial remission of apatient having AS comprising administering to the patient a TNFαinhibitor.

The invention provides a method for monitoring the effectiveness of aTNFα inhibitor for the treatment of ankylosing spondylitis (AS) in ahuman subject comprising using a mean baseline AS Quality of LifeQuestionnaire (ASQoL) of a patient population having AS and a mean ASQoLscore of the patient population following administration of the TNFαinhibitor, wherein a mean decrease in the ASQoL score of at least about3 indicates that the TNFα inhibitor is effective at treating AS. In oneembodiment, the TNFα inhibitor has already been administered to thepre-selected patient population.

The invention also provides a method for monitoring the effectiveness ofa TNFα inhibitor for the treatment of ankylosing spondylitis (AS) in ahuman subject comprising using a mean baseline Maastricht AS EnthesitisScore (MASES) of a patient population having AS and a mean MASES scoreof the patient population following administration of the TNFαinhibitor, wherein a mean decrease in the MASES score of at least about2 indicates that the TNFα inhibitor is effective at treating AS. In oneembodiment, the TNFα inhibitor has already been administered to thepre-selected patient population.

The invention also provides a method of testing the effectiveness of aTNFα inhibitor for the treatment of ankylosing spondylitis (AS)comprising using a mean baseline BASDAI score of a preselected patientpopulation having AS and a mean BASDAI score of the patient populationfollowing administration of the TNFα inhibitor, wherein a BASDAI20response rate in at least about 70% of the patient population indicatesthe TNFα inhibitor is effective for the treatment of AS.

The invention further provides a method of testing the effectiveness ofa TNFα inhibitor for the treatment of ankylosing spondylitis (AS)comprising using a mean baseline BASDAI score of a preselected patientpopulation having AS and a mean BASDAI score of the patient populationfollowing administration of the TNFα inhibitor, wherein a BASDAI70response rate in at least about 25% of the patient population indicatesthe TNFα inhibitor is effective for the treatment of AS.

In one embodiment, the patient population has a mean BASDAI of about6.6.

In another embodiment, the TNFα inhibitor is selected from the groupconsisting of a TNFα antibody, or an antigen-binding portion thereof, aTNF fusion protein, or a recombinant TNF binding protein.

In another embodiment, the TNF fusion protein is etanercept.

In still another embodiment, the TNFα antibody, or antigen-bindingportion thereof, is selected from the group consisting of a chimericantibody, a humanized antibody, and a multivalent antibody. In oneembodiment, the TNFα antibody, or antigen-binding portion thereof, is ahuman antibody.

In one embodiment, the TNFα antibody, or antigen-binding portionthereof, is an isolated human antibody that dissociates from human TNFαwith a K_(d) Of 1×10⁻⁸ M or less and a K_(off) rate constant of 1×10⁻³s⁻¹ or less, both determined by surface plasmon resonance, andneutralizes human TNFα cytotoxicity in a standard in vitro L929 assaywith an IC₅₀ of 1×10⁻⁷ M or less.

In another embodiment, the TNFα antibody is an isolated human antibody,or antigen-binding portion thereof, with the following characteristics:

a) dissociates from human TNFα with a K_(off) rate constant of 1×10⁻³s⁻¹ or less, as determined by surface plasmon resonance;

b) has a light chain CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single alaninesubstitution at position 1, 4, 5, 7 or 8 or by one to five conservativeamino acid substitutions at positions 1, 3, 4, 6, 7, 8 and/or 9;

c) has a heavy chain CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single alaninesubstitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by one to fiveconservative amino acid substitutions at positions 2, 3, 4, 5, 6, 8, 9,10, 11 and/or 12.

In another embodiment, the TNFα antibody is an isolated human antibody,or an antigen binding portion thereof, with a light chain variableregion (LCVR) comprising the amino acid sequence of SEQ ID NO: 1 and aheavy chain variable region (HCVR) comprising the amino acid sequence ofSEQ ID NO: 2.

In one embodiment, the human TNFα antibody, or antigen-binding portionthereof, is adalimumab.

In one embodiment, the TNFα antibody, or antigen-binding portionthereof, is a 40 mg dose.

In another embodiment, the TNFα antibody, or antigen-binding portionthereof, is administered subcutaneously.

In still another embodiment, the TNFα antibody, or antigen-bindingportion thereof, is infliximab or golimumab.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 graphically depicts the improvement of BASDAI20, 50 and 70 after2, 12 and 52 weeks of treatment with adalimumab.

FIG. 2 shows the improvement of ASAS20, 40 and 70 criteria after 2, 12and 52 weeks of treatment with adalimumab.

FIG. 3 describes an overview of the Canadian AS study design. The dashedline indicates the early escape option at Weeks 22, 16, and 20.

FIG. 4 shows the change in mean FACIT-Fatigue score by treatment groupover time.

FIG. 5 shows the change in mean BASDAI-fatigue item score by treatmentgroup over time.

FIG. 6 shows the mean SF-36 vitality domain score by treatment groupover time.

FIG. 7 outlines the Study H design. The dashed line indicates the earlyescape option at Weeks 22, 16, and 20.

FIG. 8 graphically depicts the ASA40 response through Week 24. ***Statistically significant at p=0.001 level, wherein the p value is fromPearson's Chi-square test. ASAS40 values are imputed.

FIG. 9 depicts the ASA20 response for those patients who switched to EETat Week 12 of the Canadian AS study. Data includes patients who enteredearly-escape open-label therapy at Week 14.

FIG. 10 depicts the ASAS20 response time course observed in patientsfrom the Canadian AS study. Observed analysis excludes patients withmissing data, but includes patients who remained on randomized studydrug or who elected early escape treatment. A statistically significantdifference was present between adalimumab and placebo treatment groupsat the p=0.01 level.

FIG. 11 graphically depicts the time course of ASAS 20 (Imputed) inpatients enrolled in Study H. Patients who received early escape therapyor discontinued are counted as non-responders at Weeks 12 and 24.***Statistically significant at p=0.001 level.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The term “human TNFα” (abbreviated herein as hTNFα, or simply hTNF), asused herein, is intended to refer to a human cytokine that exists as a17 kD secreted form and a 26 kD membrane associated form, thebiologically active form of which is composed of a trimer ofnoncovalently bound 17 kD molecules. The structure of hTNFα is describedfurther in, for example, Pennica, D., et al. (1984) Nature 312:724-729;Davis, J. M., et al. (1987) Biochemistry 26:1322-1326; and Jones, E. Y.,et al. (1989) Nature 338:225-228. The term human TNFα is intended toinclude recombinant human TNFα (rhTNFα), which can be prepared bystandard recombinant expression methods or purchased commercially (R & DSystems, Catalog No. 210-TA, Minneapolis, Minn.). TNFα is also referredto as TNF.

The term “TNFα inhibitor” includes agents which interfere with TNFαactivity. The term also includes each of the anti-TNFα human antibodiesand antibody portions described herein as well as those described inU.S. Pat. Nos. 6,090,382; 6,258,562; 6,509,015, and in U.S. patentapplication Ser. Nos. 09/801,185 and 10/302,356. In one embodiment, theTNFα inhibitor used in the invention is an anti-TNFα antibody, or afragment thereof, including infliximab (Remicade®, Johnson and Johnson;described in U.S. Pat. No. 5,656,272, incorporated by reference herein),CDP571 (a humanized monoclonal anti-TNF-alpha IgG4 antibody), CDP 870 (ahumanized monoclonal anti-TNF-alpha antibody fragment), an anti-TNF dAb(Peptech), CNTO 148 (golimumab; Medarex and Centocor, see WO 02/12502),and adalimumab (HUMIRA® Abbott Laboratories, a human anti-TNF mAb,described in U.S. Pat. No. 6,090,382 as D2E7). Additional TNF antibodieswhich may be used in the invention are described in U.S. Pat. Nos.6,593,458; 6,498,237; 6,451,983; and 6,448,380, each of which isincorporated by reference herein. In another embodiment, the TNFαinhibitor is a TNF fusion protein, e.g., etanercept (Enbrel®, Amgen;described in WO 91/03553 and WO 09/406,476, incorporated by referenceherein). In another embodiment, the TNFα inhibitor is a recombinant TNFbinding protein (r-TBP-I) (Serono).

The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules comprised of four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds.Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as HCVR or VH) and a heavy chain constant region.The heavy chain constant region is comprised of three domains, CH1, CH2and CH3. Each light chain is comprised of a light chain variable region(abbreviated herein as LCVR or VL) and a light chain constant region.The light chain constant region is comprised of one domain, CL. The VHand VL regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each VH and VL is composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The antibodies of the inventionare described in further detail in U.S. Pat. Nos. 6,090,382; 6,258,562;and 6,509,015, each of which is incorporated herein by reference in itsentirety.

The term “antigen-binding portion” or “antigen-binding fragment” of anantibody (or simply “antibody portion”), as used herein, refers to oneor more fragments of an antibody that retain the ability to specificallybind to an antigen (e.g., hTNFα). It has been shown that theantigen-binding function of an antibody can be performed by fragments ofa full-length antibody. Binding fragments include Fab, Fab′, F(ab′)₂,Fabc, Fv, single chains, and single-chain antibodies. Examples ofbinding fragments encompassed within the term “antigen-binding portion”of an antibody include (i) a Fab fragment, a monovalent fragmentconsisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)₂ fragment, abivalent fragment comprising two Fab fragments linked by a disulfidebridge at the hinge region; (iii) a Fd fragment consisting of the VH andCH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, (v) a dAb fragment (Ward et al. (1989) Nature341:544-546), which consists of a VH or VL domain; and (vi) an isolatedcomplementarity determining region (CDR). Furthermore, although the twodomains of the Fv fragment, VL and VH, are coded for by separate genes,they can be joined, using recombinant methods, by a synthetic linkerthat enables them to be made as a single protein chain in which the VLand VH regions pair to form monovalent molecules (known as single chainFv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Hustonet al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such singlechain antibodies are also intended to be encompassed within the term“antigen-binding portion” of an antibody. Other forms of single chainantibodies, such as diabodies are also encompassed. Diabodies arebivalent, bispecific antibodies in which VH and VL domains are expressedon a single polypeptide chain, but using a linker that is too short toallow for pairing between the two domains on the same chain, therebyforcing the domains to pair with complementary domains of another chainand creating two antigen binding sites (see e.g., Holliger et al. (1993)Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak et al. (1994) Structure2:1121-1123). The antibody portions of the invention are described infurther detail in U.S. Pat. Nos. 6,090,382, 6,258,562, 6,509,015, eachof which is incorporated herein by reference in its entirety.

Still further, an antibody or antigen-binding portion thereof may bepart of a larger immunoadhesion molecules, formed by covalent ornoncovalent association of the antibody or antibody portion with one ormore other proteins or peptides. Examples of such immunoadhesionmolecules include use of the streptavidin core region to make atetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) HumanAntibodies and Hybridomas 6:93-101) and use of a cysteine residue, amarker peptide and a C-terminal polyhistidine tag to make bivalent andbiotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol.Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab′)₂fragments, can be prepared from whole antibodies using conventionaltechniques, such as papain or pepsin digestion, respectively, of wholeantibodies. Moreover, antibodies, antibody portions and immunoadhesionmolecules can be obtained using standard recombinant DNA techniques, asdescribed herein.

A “conservative amino acid substitution”, as used herein, is one inwhich one amino acid residue is replaced with another amino acid residuehaving a similar side chain. Families of amino acid residues havingsimilar side chains have been defined in the art, including basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine).

“Chimeric antibodies” refers to antibodies wherein one portion of eachof the amino acid sequences of heavy and light chains is homologous tocorresponding sequences in antibodies derived from a particular speciesor belonging to a particular class, while the remaining segment of thechains is homologous to corresponding sequences from another species. Inone embodiment, the invention features a chimeric antibody orantigen-binding fragment, in which the variable regions of both lightand heavy chains mimics the variable regions of antibodies derived fromone species of mammals, while the constant portions are homologous tothe sequences in antibodies derived from another species. In a preferredembodiment of the invention, chimeric antibodies are made by graftingCDRs from a mouse antibody onto the framework regions of a humanantibody.

“Humanized antibodies” refer to antibodies which comprise at least onechain comprising variable region framework residues substantially from ahuman antibody chain (referred to as the acceptor immunoglobulin orantibody) and at least one complementarity determining region (CDR)substantially from a non-human-antibody (e.g., mouse). In addition tothe grafting of the CDRs, humanized antibodies typically undergo furtheralterations in order to improve affinity and/or immunogenicity.

The term “multivalent antibody” refers to an antibody comprising morethan one antigen recognition site. For example, a “bivalent” antibodyhas two antigen recognition sites, whereas a “tetravalent” antibody hasfour antigen recognition sites. The terms “monospecific”, “bispecific”,“trispecific”, “tetraspecific”, etc. refer to the number of differentantigen recognition site specificities (as opposed to the number ofantigen recognition sites) present in a multivalent antibody. Forexample, a “monospecific” antibody's antigen recognition sites all bindthe same epitope. A “bispecific” or “dual specific” antibody has atleast one antigen recognition site that binds a first epitope and atleast one antigen recognition site that binds a second epitope that isdifferent from the first epitope. A “multivalent monospecific” antibodyhas multiple antigen recognition sites that all bind the same epitope. A“multivalent bispecific” antibody has multiple antigen recognitionsites, some number of which bind a first epitope and some number ofwhich bind a second epitope that is different from the first epitope

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs and in particular CDR3. However, the term “humanantibody”, as used herein, is not intended to include antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell (describedfurther below), antibodies isolated from a recombinant, combinatorialhuman antibody library (described further below), antibodies isolatedfrom an animal (e.g., a mouse) that is transgenic for humanimmunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res.20:6287) or antibodies prepared, expressed, created or isolated by anyother means that involves splicing of human immunoglobulin genesequences to other DNA sequences. Such recombinant human antibodies havevariable and constant regions derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies are subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the VH and VL regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline VH and VL sequences, may not naturally existwithin the human antibody germline repertoire in vivo.

Such chimeric, humanized, human, and dual specific antibodies can beproduced by recombinant DNA techniques known in the art, for exampleusing methods described in PCT International Application No.PCT/US86/02269; European Patent Application No. 184,187; European PatentApplication No. 171,496; European Patent Application No. 173,494; PCTInternational Publication No. WO 86/01533; U.S. Pat. No. 4,816,567;European Patent Application No. 125,023; Better et al. (1988) Science240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al.(1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987)Cancer Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; Shaw etal. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison (1985) Science229:1202-1207; Oi et al. (1986) BioTechniques 4:214; U.S. Pat. No.5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al.(1988) Science 239:1534; and Beidler et al. (1988) J. Immunol.141:4053-4060, Queen et al., Proc. Natl. Acad. Sci. USA 86:10029-10033(1989), U.S. Pat. No. 5,530,101, U.S. Pat. No. 5,585,089, U.S. Pat. No.5,693,761, U.S. Pat. No. 5,693,762, Selick et al., WO 90/07861, andWinter, U.S. Pat. No. 5,225,539.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds hTNFα is substantially free of antibodies that specifically bindantigens other than hTNFα). An isolated antibody that specifically bindshTNFα may, however, have cross-reactivity to other antigens, such asTNFα molecules from other species. Moreover, an isolated antibody may besubstantially free of other cellular material and/or chemicals.

A “neutralizing antibody”, as used herein (or an “antibody thatneutralized hTNFα activity”), is intended to refer to an antibody whosebinding to hTNFα results in inhibition of the biological activity ofhTNFα. This inhibition of the biological activity of hTNFα can beassessed by measuring one or more indicators of hTNFα biologicalactivity, such as hTNFα-induced cytotoxicity (either in vitro or invivo), hTNFαα-induced cellular activation and hTNFα binding to hTNFαreceptors. These indicators of hTNFα biological activity can be assessedby one or more of several standard in vitro or in vivo assays known inthe art (see U.S. Pat. No. 6,090,382). Preferably, the ability of anantibody to neutralize hTNFα activity is assessed by inhibition ofhTNFα-induced cytotoxicity of L929 cells. As an additional oralternative parameter of hTNFα activity, the ability of an antibody toinhibit hTNFα-induced expression of ELAM-1 on HUVEC, as a measure ofhTNFα-induced cellular activation, can be assessed.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIAcore system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). Forfurther descriptions, see Example 1 of U.S. Pat. No. 6,258,562 andJönsson et al. (1993) Ann. Biol. Clin. 51:19; Jönsson et al. (1991)Biotechniques 11:620-627; Johnsson et al. (1995) J. Mol. Recognit.8:125; and Johnnson et al. (1991) Anal. Biochem. 198:268.

The term “K_(off)”, as used herein, is intended to refer to the off rateconstant for dissociation of an antibody from the antibody/antigencomplex.

The term “K_(d)”, as used herein, is intended to refer to thedissociation constant of a particular antibody-antigen interaction.

The term “IC₅₀” as used herein, is intended to refer to theconcentration of the inhibitor required to inhibit the biologicalendpoint of interest, e.g., neutralize cytotoxicity activity.

The term “dose,” as used herein, refers to an amount of TNFα inhibitorwhich is administered to a subject.

The term “dosing”, as used herein, refers to the administration of asubstance (e.g., an anti-TNFα antibody) to achieve a therapeuticobjective (e.g., treatment of ankylosing spondylitis (AS)).

A “dosing regimen” describes a treatment schedule for a TNFα inhibitor,e.g., a treatment schedule over a prolonged period of time and/orthroughout the course of treatment, e.g. administering a first dose of aTNFα inhibitor at week 0 followed by a second dose of a TNFα inhibitoron a biweekly dosing regimen.

The terms “biweekly dosing regimen”, “biweekly dosing”, and “biweeklyadministration”, as used herein, refer to the time course ofadministering a substance (e.g., an anti-TNFα antibody) to a subject toachieve a therapeutic objective, e.g, throughout the course oftreatment. The biweekly dosing regimen is not intended to include aweekly dosing regimen. Preferably, the substance is administered every9-19 days, more preferably, every 11-17 days, even more preferably,every 13-15 days, and most preferably, every 14 days. In one embodiment,the biweekly dosing regimen is initiated in a subject at week 0 oftreatment. In one embodiment, biweekly dosing includes a dosing regimenwherein doses of a TNFα inhibitor are administered to a subject everyother week beginning at week 0. In one embodiment, biweekly dosingincludes a dosing regimen where doses of a TNFα inhibitor areadministered to a subject every other week consecutively for a giventime period, e.g., 4 weeks, 8 weeks, 16, weeks, 24 weeks, etc. Biweeklydosing methods are also described in US 20030235585, incorporated byreference herein.

The term “combination” as in the phrase “a first agent in combinationwith a second agent” includes co-administration of a first agent and asecond agent, which for example may be dissolved or intermixed in thesame pharmaceutically acceptable carrier, or administration of a firstagent, followed by the second agent, or administration of the secondagent, followed by the first agent. The present invention, therefore,includes methods of combination therapeutic treatment and combinationpharmaceutical compositions. In one embodiment, a TNFα antibody isadministered in combination with methotrexate for the treatment ofankylosing spondylitis.

The term “concomitant” as in the phrase “concomitant therapeutictreatment” includes administering an agent in the presence of a secondagent. A concomitant therapeutic treatment method includes methods inwhich the first, second, third, or additional agents areco-administered. A concomitant therapeutic treatment method alsoincludes methods in which the first or additional agents areadministered in the presence of a second or additional agents, whereinthe second or additional agents, for example, may have been previouslyadministered. A concomitant therapeutic treatment method may be executedstep-wise by different actors. For example, one actor may administer toa subject a first agent and a second actor may to administer to thesubject a second agent, and the administering steps may be executed atthe same time, or nearly the same time, or at distant times, so long asthe first agent (and additional agents) are after administration in thepresence of the second agent (and additional agents). The actor and thesubject may be the same entity (e.g., human).

The term “combination therapy”, as used herein, refers to theadministration of two or more therapeutic substances, e.g., an anti-TNFαantibody and another drug. The other drug(s) may be administeredconcomitant with, prior to, or following the administration of ananti-TNFα antibody.

The term “treatment,” as used within the context of the presentinvention, is meant to include therapeutic treatment, as well asprophylactic or suppressive measures, for the treatment of ankylosingspondylitis. For example, the term treatment may include administrationof a TNFα inhibitor prior to or following the onset of ankylosingspondylitis thereby preventing or removing signs of the disease ordisorder. As another example, administration of a TNFα inhibitor afterclinical manifestation of ankylosing spondylitis to combat the symptomsand/or complications and disorders associated with ankylosingspondylitis comprises “treatment” of the disease. Further,administration of the agent after onset and after clinical symptomsand/or complications have developed where administration affectsclinical parameters of the disease or disorder and perhaps ameliorationof the disease, comprises “treatment” of the ankylosing spondylitis.

Those “in need of treatment” include mammals, such as humans, alreadyhaving AS, including those in which the disease or disorder is to beprevented.

The invention provides improved uses and compositions for treatingankylosing spondylitis with a TNFα inhibitor, e.g., a human TNFαantibody, or an antigen-binding portion thereof. Compositions andarticles of manufacture, including kits, relating to the methods anduses for treating ankylosing spondylitis are also contemplated as partof the invention. Various aspects of the invention are described infurther detail herein.

II. TNF Inhibitors

A TNFα inhibitor which is used in the methods and compositions of theinvention includes any agent which interferes with TNFα activity. In apreferred embodiment, the TNFα inhibitor can neutralize TNFα activity,particularly detrimental TNFα activity which is associated withankylosing spondylitis, and related complications and symptoms.

In one embodiment, the TNFα inhibitor used in the invention is an TNFαantibody (also referred to herein as a TNFα antibody), or anantigen-binding fragment thereof, including chimeric, humanized, andhuman antibodies. Examples of TNFα antibodies which may be used in theinvention include, but not limited to, infliximab (Remicade®, Johnsonand Johnson; described in U.S. Pat. No. 5,656,272, incorporated byreference herein), CDP571 (a humanized monoclonal anti-TNF-alpha IgG4antibody), CDP 870 (a humanized monoclonal anti-TNF-alpha antibodyfragment), an anti-TNF dAb (Peptech), CNTO 148 (golimumab; Medarex andCentocor, see WO 02/12502), and adalimumab (HUMIRA® Abbott Laboratories,a human anti-TNF mAb, described in U.S. Pat. No. 6,090,382 as D2E7).Additional TNF antibodies which may be used in the invention aredescribed in U.S. Pat. Nos. 6,593,458; 6,498,237; 6,451,983; and6,448,380, each of which is incorporated by reference herein.

Other examples of TNFα inhibitors which may be used in the methods andcompositions of the invention include etanercept (Enbrel, described inWO 91/03553 and WO 09/406476), soluble TNF receptor Type I, a pegylatedsoluble TNF receptor Type I (PEGs TNF-R1), p55TNFR1gG (Lenercept), andrecombinant TNF binding protein (r-TBP-I) (Serono).

In one embodiment, the term “TNFα inhibitor” excludes infliximab. In oneembodiment, the term “TNFα inhibitor” excludes adalimumab. In anotherembodiment, the term “TNFα inhibitor” excludes adalimumab andinfliximab.

In one embodiment, the term “TNFα inhibitor” excludes etanercept, and,optionally, adalimumab, infliximab, or adalimumab and infliximab.

In one embodiment, the term “TNFα antibody” excludes infliximab. In oneembodiment, the term “TNFα antibody” excludes adalimumab. In anotherembodiment, the term “TNFα antibody” excludes adalimumab and infliximab.

In one embodiment, the invention features uses and composition fortreating or determining the efficacy of a TNFα inhibitor for thetreatment of ankylosing spondylitis, wherein the TNFα antibody is anisolated human antibody, or antigen-binding portion thereof, that bindsto human TNFα with high affinity and a low off rate, and also has a highneutralizing capacity. Preferably, the human antibodies used in theinvention are recombinant, neutralizing human anti-hTNFα antibodies. Themost preferred recombinant, neutralizing antibody of the invention isreferred to herein as D2E7, also referred to as HUMIRA® or adalimumab(the amino acid sequence of the D2E7 VL region is shown in SEQ ID NO: 1;the amino acid sequence of the D2E7 VH region is shown in SEQ ID NO: 2).The properties of D2E7 (adalimumab/HUMIRA®) have been described inSalfeld et al., U.S. Pat. Nos. 6,090,382, 6,258,562, and 6,509,015,which are each incorporated by reference herein. The methods of theinvention may also be performed using chimeric and humanized murineanti-hTNFα antibodies which have undergone clinical testing fortreatment of rheumatoid arthritis (see e.g., Elliott, M. J., et al.(1994) Lancet 344:1125-1127; Elliot, M. J., et al. (1994) Lancet344:1105-1110; Rankin, E. C., et al. (1995) Br. J. Rheumatol.34:334-342).

In one embodiment, the method of the invention includes determining theefficacy of a TNFα inhibitor, including human antibodies and antibodyportions with equivalent properties to D2E7, such as high affinitybinding to hTNFα with low dissociation kinetics and high neutralizingcapacity, for the treatment of ankylosing spondylitis. In oneembodiment, the invention provides treatment with an isolated humanantibody, or an antigen-binding portion thereof, that dissociates fromhuman TNFα with a K_(d) of 1×10⁻⁸ M or less and a K_(off) rate constantof 1×10⁻³ s⁻¹ or less, both determined by surface plasmon resonance, andneutralizes human TNFα cytotoxicity in a standard in vitro L929 assaywith an IC₅₀ Of 1×10⁻⁷ M or less. More preferably, the isolated humanantibody, or antigen-binding portion thereof, dissociates from humanTNFα with a K_(off) of 5×10⁻⁴ s⁻¹ or less, or even more preferably, witha K_(off) of 1×10⁻⁴ s⁻¹ or less. More preferably, the isolated humanantibody, or antigen-binding portion thereof, neutralizes human TNFαcytotoxicity in a standard in vitro L929 assay with an IC₅₀ of 1×10⁻⁸ Mor less, even more preferably with an IC₅₀ of 1×10⁻⁹ M or less and stillmore preferably with an IC₅₀ of 1×10⁻¹⁰ M or less. In a preferredembodiment, the antibody is an isolated human recombinant antibody, oran antigen-binding portion thereof.

It is well known in the art that antibody heavy and light chain CDR3domains play an important role in the binding specificity/affinity of anantibody for an antigen. Accordingly, in another aspect, the inventionpertains to treating AS by administering human antibodies that have slowdissociation kinetics for association with hTNFα and that have light andheavy chain CDR3 domains that structurally are identical to or relatedto those of D2E7. Position 9 of the D2E7 VL CDR3 can be occupied by Alaor Thr without substantially affecting the K_(off). Accordingly, aconsensus motif for the D2E7 VL CDR3 comprises the amino acid sequence:Q-R-Y-N-R-A-P-Y-(T/A) (SEQ ID NO: 3). Additionally, position 12 of theD2E7 VH CDR3 can be occupied by Tyr or Asn, without substantiallyaffecting the K_(off). Accordingly, a consensus motif for the D2E7 VHCDR3 comprises the amino acid sequence: V-S-Y-L-S-T-A-S-S-L-D-(Y/N) (SEQID NO: 4). Moreover, as demonstrated in Example 2 of U.S. Pat. No.6,090,382, the CDR3 domain of the D2E7 heavy and light chains isamenable to substitution with a single alanine residue (at position 1,4, 5, 7 or 8 within the VL CDR3 or at position 2, 3, 4, 5, 6, 8, 9, 10or 11 within the VH CDR3) without substantially affecting the K_(off).Still further, the skilled artisan will appreciate that, given theamenability of the D2E7 VL and VH CDR3 domains to substitutions byalanine, substitution of other amino acids within the CDR3 domains maybe possible while still retaining the low off rate constant of theantibody, in particular substitutions with conservative amino acids.Preferably, no more than one to five conservative amino acidsubstitutions are made within the D2E7 VL and/or VH CDR3 domains. Morepreferably, no more than one to three conservative amino acidsubstitutions are made within the D2E7 VL and/or VH CDR3 domains.Additionally, conservative amino acid substitutions should not be madeat amino acid positions critical for binding to hTNFα. Positions 2 and 5of the D2E7 VL CDR3 and positions 1 and 7 of the D2E7 VH CDR3 appear tobe critical for interaction with hTNFα and thus, conservative amino acidsubstitutions preferably are not made at these positions (although analanine substitution at position 5 of the D2E7 VL CDR3 is acceptable, asdescribed above) (see U.S. Pat. No. 6,090,382).

Accordingly, in another embodiment, the antibody or antigen-bindingportion thereof preferably contains the following characteristics:

a) dissociates from human TNFα with a K_(off) rate constant of 1×10⁻³s⁻¹ or less, as determined by surface plasmon resonance;

b) has a light chain CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single alaninesubstitution at position 1, 4, 5, 7 or 8 or by one to five conservativeamino acid substitutions at positions 1, 3, 4, 6, 7, 8 and/or 9;

c) has a heavy chain CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single alaninesubstitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by one to fiveconservative amino acid substitutions at positions 2, 3, 4, 5, 6, 8, 9,10, 11 and/or 12.

More preferably, the antibody, or antigen-binding portion thereof,dissociates from human TNFα with a K_(off) of 5×10⁻⁴ s⁻¹ or less. Evenmore preferably, the antibody, or antigen-binding portion thereof,dissociates from human TNFα with a K_(off) of 1×10⁻⁴ s⁻¹ or less.

In yet another embodiment, the antibody or antigen-binding portionthereof preferably contains a light chain variable region (LCVR) havinga CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3, ormodified from SEQ ID NO: 3 by a single alanine substitution at position1, 4, 5, 7 or 8, and with a heavy chain variable region (HCVR) having aCDR3 domain comprising the amino acid sequence of SEQ ID NO: 4, ormodified from SEQ ID NO: 4 by a single alanine substitution at position2, 3, 4, 5, 6, 8, 9, 10 or 11. Preferably, the LCVR further has a CDR2domain comprising the amino acid sequence of SEQ ID NO: 5 (i.e., theD2E7 VL CDR2) and the HCVR further has a CDR2 domain comprising theamino acid sequence of SEQ ID NO: 6 (i.e., the D2E7 VH CDR2). Even morepreferably, the LCVR further has CDR1 domain comprising the amino acidsequence of SEQ ID NO: 7 (i.e., the D2E7 VL CDR1) and the HCVR has aCDR1 domain comprising the amino acid sequence of SEQ ID NO: 8 (i.e.,the D2E7 VH CDR1). The framework regions for VL preferably are from theV_(κ)I human germline family, more preferably from the A20 humangermline Vk gene and most preferably from the D2E7 VL frameworksequences shown in FIGS. 1A and 1B of U.S. Pat. No. 6,090,382. Theframework regions for VH preferably are from the V_(H)3 human germlinefamily, more preferably from the DP-31 human germline VH gene and mostpreferably from the D2E7 VH framework sequences shown in FIGS. 2A and 2Bof U.S. Pat. No. 6,090,382.

Accordingly, in another embodiment, the antibody or antigen-bindingportion thereof preferably contains a light chain variable region (LCVR)comprising the amino acid sequence of SEQ ID NO: 1 (i.e., the D2E7 VL)and a heavy chain variable region (HCVR) comprising the amino acidsequence of SEQ ID NO: 2 (i.e., the D2E7 VH). In certain embodiments,the antibody comprises a heavy chain constant region, such as an IgG1,IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region. Preferably, theheavy chain constant region is an IgG1 heavy chain constant region or anIgG4 heavy chain constant region. Furthermore, the antibody can comprisea light chain constant region, either a kappa light chain constantregion or a lambda light chain constant region. Preferably, the antibodycomprises a kappa light chain constant region. Alternatively, theantibody portion can be, for example, a Fab fragment or a single chainFv fragment.

In still other embodiments, the invention includes uses of an isolatedhuman antibody, or an antigen-binding portions thereof, containingD2E7-related VL and VH CDR3 domains. For example, antibodies, orantigen-binding portions thereof, with a light chain variable region(LCVR) having a CDR3 domain comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 12,SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ IDNO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 orwith a heavy chain variable region (HCVR) having a CDR3 domaincomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 4, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO:30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 and SEQID NO: 35.

The TNFα antibody used in the methods and compositions of the inventionmay be modified for improved treatment of ankylosing spondylitis. Insome embodiments, the TNFα antibody or antigen binding fragmentsthereof, is chemically modified to provide a desired effect. Forexample, pegylation of antibodies and antibody fragments of theinvention may be carried out by any of the pegylation reactions known inthe art, as described, for example, in the following references: Focuson Growth Factors 3:4-10 (1992); EP 0 154 316; and EP 0 401 384 (each ofwhich is incorporated by reference herein in its entirety). Preferably,the pegylation is carried out via an acylation reaction or an alkylationreaction with a reactive polyethylene glycol molecule (or an analogousreactive water-soluble polymer). A preferred water-soluble polymer forpegylation of the antibodies and antibody fragments of the invention ispolyethylene glycol (PEG). As used herein, “polyethylene glycol” ismeant to encompass any of the forms of PEG that have been used toderivatize other proteins, such as mono (Cl-ClO) alkoxy- oraryloxy-polyethylene glycol.

Methods for preparing pegylated antibodies and antibody fragments of theinvention will generally comprise the steps of (a) reacting the antibodyor antibody fragment with polyethylene glycol, such as a reactive esteror aldehyde derivative of PEG, under conditions whereby the antibody orantibody fragment becomes attached to one or more PEG groups, and (b)obtaining the reaction products. It will be apparent to one of ordinaryskill in the art to select the optimal reaction conditions or theacylation reactions based on known parameters and the desired result.

Pegylated antibodies and antibody fragments may generally be used totreat ankylosing spondylitis by administration of the TNFα antibodiesand antibody fragments described herein. Generally the pegylatedantibodies and antibody fragments have increased half-life, as comparedto the nonpegylated antibodies and antibody fragments. The pegylatedantibodies and antibody fragments may be employed alone, together, or incombination with other pharmaceutical compositions.

In yet another embodiment of the invention, TNFα antibodies or fragmentsthereof can be altered wherein the constant region of the antibody ismodified to reduce at least one constant region-mediated biologicaleffector function relative to an unmodified antibody. To modify anantibody of the invention such that it exhibits reduced binding to theFc receptor, the immunoglobulin constant region segment of the antibodycan be mutated at particular regions necessary for Fc receptor (FcR)interactions (see e.g., Canfield, S. M. and S. L. Morrison (1991) J.Exp. Med. 173:1483-1491; and Lund, J. et al. (1991) J. of Immunol.147:2657-2662). Reduction in FcR binding ability of the antibody mayalso reduce other effector functions which rely on FcR interactions,such as opsonization and phagocytosis and antigen-dependent cellularcytotoxicity.

An antibody or antibody portion used in the methods of the invention canbe derivatized or linked to another functional molecule (e.g., anotherpeptide or protein). Accordingly, the antibodies and antibody portionsof the invention are intended to include derivatized and otherwisemodified forms of the human anti-hTNFα antibodies described herein,including immunoadhesion molecules. For example, an antibody or antibodyportion of the invention can be functionally linked (by chemicalcoupling, genetic fusion, noncovalent association or otherwise) to oneor more other molecular entities, such as another antibody (e.g., abispecific antibody or a diabody), a detectable agent, a cytotoxicagent, a pharmaceutical agent, and/or a protein or peptide that canmediate associate of the antibody or antibody portion with anothermolecule (such as a streptavidin core region or a polyhistidine tag).

One type of derivatized antibody is produced by crosslinking two or moreantibodies (of the same type or of different types, e.g., to createbispecific antibodies). Suitable crosslinkers include those that areheterobifunctional, having two distinctly reactive groups separated byan appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimideester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkersare available from Pierce Chemical Company, Rockford, Ill.

Useful detectable agents with which an antibody or antibody portion ofthe invention may be derivatized include fluorescent compounds.Exemplary fluorescent detectable agents include fluorescein, fluoresceinisothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonylchloride, phycoerythrin and the like. An antibody may also bederivatized with detectable enzymes, such as alkaline phosphatase,horseradish peroxidase, glucose oxidase and the like. When an antibodyis derivatized with a detectable enzyme, it is detected by addingadditional reagents that the enzyme uses to produce a detectablereaction product. For example, when the detectable agent horseradishperoxidase is present, the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which isdetectable. An antibody may also be derivatized with biotin, anddetected through indirect measurement of avidin or streptavidin binding.

An antibody, or antibody portion, used in the methods and compositionsof the invention, can be prepared by recombinant expression ofimmunoglobulin light and heavy chain genes in a host cell. To express anantibody recombinantly, a host cell is transfected with one or morerecombinant expression vectors carrying DNA fragments encoding theimmunoglobulin light and heavy chains of the antibody such that thelight and heavy chains are expressed in the host cell and, preferably,secreted into the medium in which the host cells are cultured, fromwhich medium the antibodies can be recovered. Standard recombinant DNAmethodologies are used to obtain antibody heavy and light chain genes,incorporate these genes into recombinant expression vectors andintroduce the vectors into host cells, such as those described inSambrook, Fritsch and Maniatis (eds), Molecular Cloning; A LaboratoryManual, Second Edition, Cold Spring Harbor, N.Y., (1989), Ausubel, F. M.et al. (eds.) Current Protocols in Molecular Biology, Greene PublishingAssociates, (1989) and in U.S. Pat. No. 4,816,397 by Boss et al.

To express adalimumab (D2E7) or an adalimumab (D2E7)-related antibody,DNA fragments encoding the light and heavy chain variable regions arefirst obtained. These DNAs can be obtained by amplification andmodification of germline light and heavy chain variable sequences usingthe polymerase chain reaction (PCR). Germline DNA sequences for humanheavy and light chain variable region genes are known in the art (seee.g., the “Vbase” human germline sequence database; see also Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242; Tomlinson, I. M., et al. (1992) “The Repertoire of HumanGermline V_(H) Sequences Reveals about Fifty Groups of V_(H) Segmentswith Different Hypervariable Loops” J. Mol. Biol. 227:776-798; and Cox,J. P. L. et al. (1994) “A Directory of Human Germ-line V₇₈ SegmentsReveals a Strong Bias in their Usage” Eur. J. Immunol. 24:827-836; thecontents of each of which are expressly incorporated herein byreference). To obtain a DNA fragment encoding the heavy chain variableregion of D2E7, or a D2E7-related antibody, a member of the V_(H)3family of human germline VH genes is amplified by standard PCR. Mostpreferably, the DP-31 VH germline sequence is amplified. To obtain a DNAfragment encoding the light chain variable region of D2E7, or aD2E7-related antibody, a member of the V_(κ)I family of human germlineVL genes is amplified by standard PCR. Most preferably, the A20 VLgermline sequence is amplified. PCR primers suitable for use inamplifying the DP-31 germline VH and A20 germline VL sequences can bedesigned based on the nucleotide sequences disclosed in the referencescited supra, using standard methods.

Once the germline VH and VL fragments are obtained, these sequences canbe mutated to encode the D2E7 or D2E7-related amino acid sequencesdisclosed herein. The amino acid sequences encoded by the germline VHand VL DNA sequences are first compared to the D2E7 or D2E7-related VHand VL amino acid sequences to identify amino acid residues in the D2E7or D2E7-related sequence that differ from germline. Then, theappropriate nucleotides of the germline DNA sequences are mutated suchthat the mutated germline sequence encodes the D2E7 or D2E7-relatedamino acid sequence, using the genetic code to determine whichnucleotide changes should be made. Mutagenesis of the germline sequencesis carried out by standard methods, such as PCR-mediated mutagenesis (inwhich the mutated nucleotides are incorporated into the PCR primers suchthat the PCR product contains the mutations) or site-directedmutagenesis.

Moreover, it should be noted that if the “germline” sequences obtainedby PCR amplification encode amino acid differences in the frameworkregions from the true germline configuration (i.e., differences in theamplified sequence as compared to the true germline sequence, forexample as a result of somatic mutation), it may be desirable to changethese amino acid differences back to the true germline sequences (i.e.,“backmutation” of framework residues to the germline configuration).

Once DNA fragments encoding D2E7 or D2E7-related VH and VL segments areobtained (by amplification and mutagenesis of germline VH and VL genes,as described above), these DNA fragments can be further manipulated bystandard recombinant DNA techniques, for example to convert the variableregion genes to full-length antibody chain genes, to Fab fragment genesor to a scFv gene. In these manipulations, a VL- or VH-encoding DNAfragment is operatively linked to another DNA fragment encoding anotherprotein, such as an antibody constant region or a flexible linker. Theterm “operatively linked”, as used in this context, is intended to meanthat the two DNA fragments are joined such that the amino acid sequencesencoded by the two DNA fragments remain in-frame.

The isolated DNA encoding the VH region can be converted to afull-length heavy chain gene by operatively linking the VH-encoding DNAto another DNA molecule encoding heavy chain constant regions (CH1, CH2and CH3). The sequences of human heavy chain constant region genes areknown in the art (see e.g., Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242) and DNAfragments encompassing these regions can be obtained by standard PCRamplification. The heavy chain constant region can be an IgG1, IgG2,IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably isan IgG1 or IgG4 constant region. For a Fab fragment heavy chain gene,the VH-encoding DNA can be operatively linked to another DNA moleculeencoding only the heavy chain CH1 constant region.

The isolated DNA encoding the VL region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the VL-encoding DNA to another DNA molecule encodingthe light chain constant region, CL. The sequences of human light chainconstant region genes are known in the art (see e.g., Kabat, E. A., etal. (1991) Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242) and DNA fragments encompassing these regions can beobtained by standard PCR amplification. The light chain constant regioncan be a kappa or lambda constant region, but most preferably is a kappaconstant region.

To create a scFv gene, the VH- and VL-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly₄-Ser)₃, such that the VH and VLsequences can be expressed as a contiguous single-chain protein, withthe VL and VH regions joined by the flexible linker (see e.g., Bird etal. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad.Sci. USA 85:5879-5883; McCafferty et al., Nature (1990) 348:552-554).

To express the antibodies, or antibody portions used in the invention,DNAs encoding partial or full-length light and heavy chains, obtained asdescribed above, are inserted into expression vectors such that thegenes are operatively linked to transcriptional and translationalcontrol sequences. In this context, the term “operatively linked” isintended to mean that an antibody gene is ligated into a vector suchthat transcriptional and translational control sequences within thevector serve their intended function of regulating the transcription andtranslation of the antibody gene. The expression vector and expressioncontrol sequences are chosen to be compatible with the expression hostcell used. The antibody light chain gene and the antibody heavy chaingene can be inserted into separate vector or, more typically, both genesare inserted into the same expression vector. The antibody genes areinserted into the expression vector by standard methods (e.g., ligationof complementary restriction sites on the antibody gene fragment andvector, or blunt end ligation if no restriction sites are present).Prior to insertion of the D2E7 or D2E7-related light or heavy chainsequences, the expression vector may already carry antibody constantregion sequences. For example, one approach to converting the D2E7 orD2E7-related VH and VL sequences to full-length antibody genes is toinsert them into expression vectors already encoding heavy chainconstant and light chain constant regions, respectively, such that theVH segment is operatively linked to the CH segment(s) within the vectorand the VL segment is operatively linked to the CL segment within thevector. Additionally or alternatively, the recombinant expression vectorcan encode a signal peptide that facilitates secretion of the antibodychain from a host cell. The antibody chain gene can be cloned into thevector such that the signal peptide is linked in-frame to the aminoterminus of the antibody chain gene. The signal peptide can be animmunoglobulin signal peptide or a heterologous signal peptide (i.e., asignal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors of the invention carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, for example, in Goeddel; GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). It will be appreciated by those skilled in the artthat the design of the expression vector, including the selection ofregulatory sequences may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Preferred regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., theadenovirus major late promoter (AdMLP)) and polyoma. For furtherdescription of viral regulatory elements, and sequences thereof, seee.g., U.S. Pat. No. 5,168,062 by Stinski, U.S. Pat. No. 4,510,245 byBell et al. and U.S. Pat. No. 4,968,615 by Schaffner et al.

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors used in the invention may carryadditional sequences, such as sequences that regulate replication of thevector in host cells (e.g., origins of replication) and selectablemarker genes. The selectable marker gene facilitates selection of hostcells into which the vector has been introduced (see e.g., U.S. Pat.Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). Forexample, typically the selectable marker gene confers resistance todrugs, such as G418, hygromycin or methotrexate, on a host cell intowhich the vector has been introduced. Preferred selectable marker genesinclude the dihydrofolate reductase (DHFR) gene (for use in dhfr-hostcells with methotrexate selection/amplification) and the neo gene (forG418 selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. Although it is theoreticallypossible to express the antibodies of the invention in eitherprokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells, and most preferably mammalian host cells, is the mostpreferred because such eukaryotic cells, and in particular mammaliancells, are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active antibody. Prokaryoticexpression of antibody genes has been reported to be ineffective forproduction of high yields of active antibody (Boss, M. A. and Wood, C.R. (1985) Immunology Today 6:12-13).

Preferred mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (includingdhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad.Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., asdescribed in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol.159:601-621), NSO myeloma cells, COS cells and SP2 cells. Whenrecombinant expression vectors encoding antibody genes are introducedinto mammalian host cells, the antibodies are produced by culturing thehost cells for a period of time sufficient to allow for expression ofthe antibody in the host cells or, more preferably, secretion of theantibody into the culture medium in which the host cells are grown.Antibodies can be recovered from the culture medium using standardprotein purification methods.

Host cells can also be used to produce portions of intact antibodies,such as Fab fragments or scFv molecules. It is understood thatvariations on the above procedure are within the scope of the presentinvention. For example, it may be desirable to transfect a host cellwith DNA encoding either the light chain or the heavy chain (but notboth) of an antibody of this invention. Recombinant DNA technology mayalso be used to remove some or all of the DNA encoding either or both ofthe light and heavy chains that is not necessary for binding to hTNFα.The molecules expressed from such truncated DNA molecules are alsoencompassed by the antibodies of the invention. In addition,bifunctional antibodies may be produced in which one heavy and one lightchain are an antibody of the invention and the other heavy and lightchain are specific for an antigen other than hTNFα by crosslinking anantibody of the invention to a second antibody by standard chemicalcrosslinking methods.

In a preferred system for recombinant expression of an antibody, orantigen-binding portion thereof, of the invention, a recombinantexpression vector encoding both the antibody heavy chain and theantibody light chain is introduced into dhfr-CHO cells by calciumphosphate-mediated transfection. Within the recombinant expressionvector, the antibody heavy and light chain genes are each operativelylinked to CMV enhancer/AdMLP promoter regulatory elements to drive highlevels of transcription of the genes. The recombinant expression vectoralso carries a DHFR gene, which allows for selection of CHO cells thathave been transfected with the vector using methotrexateselection/amplification. The selected transformant host cells areculture to allow for expression of the antibody heavy and light chainsand intact antibody is recovered from the culture medium. Standardmolecular biology techniques are used to prepare the recombinantexpression vector, transfect the host cells, select for transformants,culture the host cells and recover the antibody from the culture medium.

In view of the foregoing, nucleic acid, vector and host cellcompositions that can be used for recombinant expression of theantibodies and antibody portions used in the invention include nucleicacids, and vectors comprising said nucleic acids, comprising the humanTNFα antibody adalimumab (D2E7). The nucleotide sequence encoding theD2E7 light chain variable region is shown in SEQ ID NO: 36. The CDR1domain of the LCVR encompasses nucleotides 70-102, the CDR2 domainencompasses nucleotides 148-168 and the CDR3 domain encompassesnucleotides 265-291. The nucleotide sequence encoding the D2E7 heavychain variable region is shown in SEQ ID NO: 37. The CDR1 domain of theHCVR encompasses nucleotides 91-105, the CDR2 domain encompassesnucleotides 148-198 and the CDR3 domain encompasses nucleotides 295-330.It will be appreciated by the skilled artisan that nucleotide sequencesencoding D2E7-related antibodies, or portions thereof (e.g., a CDRdomain, such as a CDR3 domain), can be derived from the nucleotidesequences encoding the D2E7 LCVR and HCVR using the genetic code andstandard molecular biology techniques.

Recombinant human antibodies of the invention in addition to D2E7 or anantigen binding portion thereof, or D2E7-related antibodies disclosedherein can be isolated by screening of a recombinant combinatorialantibody library, preferably a scFv phage display library, preparedusing human VL and VH cDNAs prepared from mRNA derived from humanlymphocytes. Methodologies for preparing and screening such librariesare known in the art. In addition to commercially available kits forgenerating phage display libraries (e.g., the Pharmacia RecombinantPhage Antibody System, catalog no. 27-9400-01; and the StratageneSurfZAP™ phage display kit, catalog no. 240612), examples of methods andreagents particularly amenable for use in generating and screeningantibody display libraries can be found in, for example, Ladner et al.U.S. Pat. No. 5,223,409; Kang et al. PCT Publication No. WO 92/18619;Dower et al. PCT Publication No. WO 91/17271; Winter et al. PCTPublication No. WO 92/20791; Markland et al. PCT Publication No. WO92/15679; Breitling et al. PCT Publication No. WO 93/01288; McCaffertyet al. PCT Publication No. WO 92/01047; Garrard et al. PCT PublicationNo. WO 92/09690; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay etal. (1992) Hum Antibod Hybridomas 3:81-65; Huse et al. (1989) Science246:1275-1281; McCafferty et al., Nature (1990) 348:552-554; Griffithset al. (1993) EMBO J. 12:725-734; Hawkins et al. (1992) J Mol Biol226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al.(1992) PNAS 89:3576-3580; Garrard et al. (1991) Bio/Technology9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; andBarbas et al. (1991) PNAS 88:7978-7982.

In a preferred embodiment, to isolate human antibodies with highaffinity and a low off rate constant for hTNFα, a murine anti-hTNFαantibody having high affinity and a low off rate constant for hTNFα(e.g., MAK 195, the hybridoma for which has deposit number ECACC 87050801) is first used to select human heavy and light chain sequenceshaving similar binding activity toward hTNFα, using the epitopeimprinting methods described in Hoogenboom et al., PCT Publication No.WO 93/06213. The antibody libraries used in this method are preferablyscFv libraries prepared and screened as described in McCafferty et al.,PCT Publication No. WO 92/01047, McCafferty et al., Nature (1990)348:552-554; and Griffiths et al., (1993) EMBO J. 12:725-734. The scFvantibody libraries preferably are screened using recombinant human TNFαas the antigen.

Once initial human VL and VH segments are selected, “mix and match”experiments, in which different pairs of the initially selected VL andVH segments are screened for hTNFα binding, are performed to selectpreferred VL/VH pair combinations. Additionally, to further improve theaffinity and/or lower the off rate constant for hTNFα binding, the VLand VH segments of the preferred VL/VH pair(s) can be randomly mutated,preferably within the CDR3 region of VH and/or VL, in a processanalogous to the in vivo somatic mutation process responsible foraffinity maturation of antibodies during a natural immune response. Thisin vitro affinity maturation can be accomplished by amplifying VH and VLregions using PCR primers complimentary to the VH CDR3 or VL CDR3,respectively, which primers have been “spiked” with a random mixture ofthe four nucleotide bases at certain positions such that the resultantPCR products encode VH and VL segments into which random mutations havebeen introduced into the VH and/or VL CDR3 regions. These randomlymutated VH and VL segments can be rescreened for binding to hTNFα andsequences that exhibit high affinity and a low off rate for hTNFαbinding can be selected.

Following screening and isolation of an anti-hTNFα antibody of theinvention from a recombinant immunoglobulin display library, nucleicacid encoding the selected antibody can be recovered from the displaypackage (e.g., from the phage genome) and subcloned into otherexpression vectors by standard recombinant DNA techniques. If desired,the nucleic acid can be further manipulated to create other antibodyforms of the invention (e.g., linked to nucleic acid encoding additionalimmunoglobulin domains, such as additional constant regions). To expressa recombinant human antibody isolated by screening of a combinatoriallibrary, the DNA encoding the antibody is cloned into a recombinantexpression vector and introduced into a mammalian host cells, asdescribed in further detail in above.

Methods of isolating human neutralizing antibodies with high affinityand a low off rate constant for hTNFα are described in U.S. Pat. Nos.6,090,382, 6,258,562, and 6,509,015, each of which is incorporated byreference herein.

Antibodies, antibody-portions, and other TNFα inhibitors for use in themethods of the invention, can be incorporated into pharmaceuticalcompositions suitable for administration to a subject. Typically, thepharmaceutical composition comprises an antibody, antibody portion, orother TNFα inhibitor, and a pharmaceutically acceptable carrier. As usedherein, “pharmaceutically acceptable carrier” includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Examples of pharmaceutically acceptablecarriers include one or more of water, saline, phosphate bufferedsaline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof. In many cases, it is preferable to includeisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride in the composition. Pharmaceuticallyacceptable carriers may further comprise minor amounts of auxiliarysubstances such as wetting or emulsifying agents, preservatives orbuffers, which enhance the shelf life or effectiveness of the antibody,antibody portion, or other TNFα inhibitor.

The compositions for use in the methods and compositions of theinvention may be in a variety of forms. These include, for example,liquid, semi-solid and solid dosage forms, such as liquid solutions(e.g., injectable and infusible solutions), dispersions or suspensions,tablets, pills, powders, liposomes and suppositories. The preferred formdepends on the intended mode of administration and therapeuticapplication. Typical preferred compositions are in the form ofinjectable or infusible solutions, such as compositions similar to thoseused for passive immunization of humans with other antibodies or otherTNFα inhibitors. The preferred mode of administration is parenteral(e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In apreferred embodiment, the antibody or other TNFα inhibitor isadministered by intravenous infusion or injection. In another preferredembodiment, the antibody or other TNFα inhibitor is administered byintramuscular or subcutaneous injection.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the active compound (i.e.,antibody, antibody portion, or other TNFα inhibitor) in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The proper fluidity of a solution can be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin.

In one embodiment, the invention includes pharmaceutical compositionscomprising an effective TNFα inhibitor and a pharmaceutically acceptablecarrier, wherein the effective TNFα inhibitor may be used to treatankylosing spondylitis.

In one embodiment, the antibody or antibody portion for use in themethods of the invention is incorporated into a pharmaceuticalformulation as described in PCT/IB03/04502 and U.S. Appln. No.20040033228, incorporated by reference herein. This formulation includesa concentration 50 mg/ml of the antibody D2E7 (adalimumab), wherein onepre-filled syringe contains 40 mg of antibody for subcutaneousinjection.

The antibodies, antibody-portions, and other TNFα inhibitors of thepresent invention can be administered by a variety of methods known inthe art, although for many therapeutic applications, the preferredroute/mode of administration is parenteral, e.g., subcutaneousinjection. In another embodiment, administration is via intravenousinjection or infusion.

As will be appreciated by the skilled artisan, the route and/or mode ofadministration will vary depending upon the desired results. In certainembodiments, the active compound may be prepared with a carrier thatwill protect the compound against rapid release, such as a controlledrelease formulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, Robinson, ed., Dekker, Inc.,New York, 1978.

In one embodiment, the TNFα antibodies and inhibitors used in theinvention are delivered to a subject subcutaneously. In one embodiment,the subject administers the TNFα inhibitor, including, but not limitedto, TNFα antibody, or antigen-binding portion thereof, tohimself/herself.

The TNFα antibodies and inhibitors used in the invention may also beadministered in the form of protein crystal formulations which include acombination of protein crystals encapsulated within a polymeric carrierto form coated particles. The coated particles of the protein crystalformulation may have a spherical morphology and be microspheres of up to500 micro meters in diameter or they may have some other morphology andbe microparticulates. The enhanced concentration of protein crystalsallows the antibody of the invention to be delivered subcutaneously. Inone embodiment, the TNFα antibodies of the invention are delivered via aprotein delivery system, wherein one or more of a protein crystalformulation or composition, is administered to a subject with aTNFα-related disorder. Compositions and methods of preparing stabilizedformulations of whole antibody crystals or antibody fragment crystalsare also described in WO 02/072636, which is incorporated by referenceherein. In one embodiment, a formulation comprising the crystallizedantibody fragments described in PCT/IB03/04502 and U.S. Appln. No.20040033228, incorporated by reference herein, are used to treatankylosing spondylitis using the treatment methods of the invention.

In certain embodiments, an antibody, antibody portion, or other TNFαinhibitor of the invention may be orally administered, for example, withan inert diluent or an assimilable edible carrier. The compound (andother ingredients, if desired) may also be enclosed in a hard or softshell gelatin capsule, compressed into tablets, or incorporated directlyinto the subject's diet. For oral therapeutic administration, thecompounds may be incorporated with excipients and used in the form ofingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. To administer a compound ofthe invention by other than parenteral administration, it may benecessary to coat the compound with, or co-administer the compound with,a material to prevent its inactivation.

Supplementary active compounds can also be incorporated into thecompositions. In certain embodiments, an antibody or antibody portionfor use in the methods of the invention is coformulated with and/orcoadministered with one or more additional therapeutic agents, includingan ankylosing spondylitis inhibitor or antagonist. For example, ananti-hTNFα antibody or antibody portion of the invention may becoformulated and/or coadministered with one or more additionalantibodies that bind other targets associated with TNFα relateddisorders (e.g., antibodies that bind other cytokines or that bind cellsurface molecules), one or more cytokines, soluble TNFα receptor (seee.g., PCT Publication No. WO 94/06476) and/or one or more chemicalagents that inhibit hTNFα production or activity (such ascyclohexane-ylidene derivatives as described in PCT Publication No. WO93/19751) or any combination thereof. Furthermore, one or moreantibodies of the invention may be used in combination with two or moreof the foregoing therapeutic agents. Such combination therapies mayadvantageously utilize lower dosages of the administered therapeuticagents, thus avoiding possible side effects, complications or low levelof response by the patient associated with the various monotherapies.

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of an antibody or antibody portion of the invention. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result. A therapeutically effective amount of the antibody,antibody portion, or other TNFα inhibitor may vary according to factorssuch as the disease state, age, sex, and weight of the individual, andthe ability of the antibody, antibody portion, other TNFα inhibitor toelicit a desired response in the individual. A therapeutically effectiveamount is also one in which any toxic or detrimental effects of theantibody, antibody portion, or other TNFα inhibitor are outweighed bythe therapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typically,since a prophylactic dose is used in subjects prior to or at an earlierstage of disease, the prophylactically effective amount will be lessthan the therapeutically effective amount.

Additional description regarding methods and uses of the inventioncomprising administration of a TNFα inhibitor are described in Part IIIof this specification, as well as the below examples.

The invention also pertains to packaged pharmaceutical compositions orkits for administering the anti-TNF antibodies of the invention for thetreatment of ankylosing spondylitis. In one embodiment of the invention,the kit comprises a TNFα inhibitor, such as an antibody and instructionsfor administration of the TNFα inhibitor for treatment of ankylosingspondylitis. The instructions may describe how, e.g., subcutaneously,and when, e.g., at week 0, week 2, week 4, etc., the different doses ofTNFα inhibitor shall be administered to a subject for treatment.

Another aspect of the invention pertains to kits containing apharmaceutical composition comprising a TNFα inhibitor, such as anantibody, and a pharmaceutically acceptable carrier and one or morepharmaceutical compositions each comprising an additional therapeuticagent useful for treating ankylosing spondylitis, and a pharmaceuticallyacceptable carrier. Alternatively, the kit comprises a singlepharmaceutical composition comprising an anti-TNFα antibody, one or moredrugs useful for treating ankylosing spondylitis, and a pharmaceuticallyacceptable carrier. The instructions may describe how, e.g.,subcutaneously, and when, e.g., at week 0, week 2, week 4, etc., thedifferent doses of TNFα inhibitor and/or the additional therapeuticagent shall be administered to a subject for treatment.

The kit may contain instructions for dosing of the pharmaceuticalcompositions for the treatment of ankylosing spondylitis. Additionaldescription regarding articles of manufacture of the invention aredescribed in subsection III.

The package or kit alternatively can contain the TNFα inhibitor and itcan be promoted for use, either within the package or throughaccompanying information, for the uses or treatment of the disordersdescribed herein. The packaged pharmaceuticals or kits further caninclude a second agent (as described herein) packaged with or copromotedwith instructions for using the second agent with a first agent (asdescribed herein).

III. Uses and Compositions for Treating Ankylosing Spondylitis

Ankylosing spondylitis (AS) is a chronic rheumatic disease. Thesacroiliac joints are affected and, to a varying degree, the spinalcolumn. The disease may also involve the peripheral joints andextra-articular structures. Patients commonly experience pain, morningstiffness and disability, all which generally increase with duration ofdisease. Systemic features, such as anorexia and fatigue may also occur.In late disease, some patients develop acute anterior uveitis,cardiovascular or pulmonary problems. Men are more commonly affectedthan women, and, as with other sponylarthritides, AS is associated withpositivity for the HLA-B27 gene.

In one embodiment, the invention provides a method for treatingankylosing spondylitis in a subject. The invention also provides amethod for achieving partial remission of a subject having AS byadministering a TNFα inhibitor.

In one embodiment, treatment of AS is achieved by administering a TNFαinhibitor to a subject in accordance with a biweekly dosing regimen.Biweekly dosing regimens can be used to treat disorders in which TNFαactivity is detrimental, and are further described in U.S. applicationSer. No. 10/163,657 (US 20030235585), incorporated by reference herein.The TNFα inhibitor, including a TNFα antibody, or an antigen-bindingportion thereof, may be administered to the subject on a biweekly dosingregimen for treatment of AS. In one embodiment, biweekly dosing includesa dosing regimen wherein doses of a TNFα inhibitor are administered to asubject every other week beginning at week 0. In one embodiment,biweekly dosing includes a dosing regimen where doses of a TNFαinhibitor are administered to a subject every other week consecutivelyfor a given time period, e.g., 4 weeks, 8 weeks, 16, weeks, 24 weeks,etc. Biweekly dosing is preferably administered parenterally, includingsubcutaneously.

In one embodiment, the human TNFα antibody, or an antigen-bindingportion thereof, is administered in a dose of about 40 mg. In oneembodiment, the human TNFα antibody, or an antigen-binding portionthereof, is adalimumab.

The invention provides a method of decreasing pain and fatigue in asubject having AS comprising administering a human TNFα antibody, orantigen-binding portion thereof, to the subject such that pain andfatigue are decreased. The invention also includes use of a human TNFαantibody, or antigen-binding portion thereof, in the manufacture of amedicament for the treatment of pain and fatigue in a subject having AS.

The invention also provides methods for improving AS in a subject basedon indices used to measure the disease state. In one embodiment, theinvention provides a method for decreasing the decrease in fatigue isdetermined by a decrease of at least about 1.9 in a BASDAI score of thesubject. In one embodiment, the decrease in fatigue is determined by adecrease of at least about 2.0 in a BASDAI score of the subject.Alternatively, improvements in fatigue in a subject having AS may bedetermined using the FACIT-F score, e.g., a meach change in FACIT scoreof about 7-8 following treatment of the subject with a TNFα inhibitor.

The invention also includes a method of inducing partial remission of ASin a subject comprising administering a human TNFα antibody, orantigen-binding portion thereof, to the subject, such that partialremission of AS is induced. The invention provides a use of a human TNFαantibody, or antigen-binding portion thereof, in the manufacture of amedicament for inducing partial remission of AS in a subject. In oneembodiment, the subject has a value of less than 20 on a scale of 0-100in all four ASAS domains.

The invention provides uses and methods for treating certainsubpopulations of AS patients with a TNFα inhibitor. Also included inthe invention are methods for determining whether a TNFα inhibitor,e.g., a TNFα antibody, or antigen-binding portion thereof, is effectivefor treating a certain subpopulation of AS patients. Thus, the inventionalso includes a method of treating a subject who is a member of asubpopulation of AS patients with a TNFα inhibitor which has beenidentified as being an effective TNFα inhibitor for the treatment of thegiven subpopulation. In one embodiment, the methods of treatmentdescribed herein may be used to treat AS subjects who have failed priortherapy with conventional drugs used to treat AS. Examples of suchconventional therapy include DMARD therapy and NSAIDs therapy. Failureon a prior therapy can be measured using any of the indices describedherein, e.g., failure to achieve an ASAS20 response.

In one embodiment, treatment of AS is achieved by administering a humanTNFα antibody, or an antigen-binding portion thereof, to a subjecthaving AS, wherein the human TNFα antibody, or an antigen-bindingportion thereof, is administered on a biweekly dosing regimen.

Methods of treatment described herein may include administration of aTNFα inhibitor to a subject to achieve a therapeutic goal, e.g.,improvement in ASAS domains, induction of partial remission, ASAS2.0,ASAS40, ASAS50, ASAS70 response, ASAS5/6 response, improvement in BASDAIscore, BASDAI20 response, BASDAI50 response, BASDAI 70 response Alsoincluded in the scope of the invention are uses of a TNFα inhibitor inthe manufacture of a medicament to achieve a therapeutic goal, e.g.,improvement in ASAS domains, induction of partial remission, ASAS20,ASAS40, ASAS50, ASAS70 response, ASAS5/6 response, improvement in BASDAIscore, BASDAI20 response, BASDAI50 response, BASDAI 70 response. Thus,where methods are described herein, it is also intended to be part ofthis invention that the use of the TNFα inhibitor in the manufacture ofa medicament for the purpose of the method is also considered within thescope of the invention. Likewise, where a use of a TNFα inhibitor in themanufacture of a medicament for the purpose of achieving a therapeuticgoal is described, methods of treatment resulting in the therapeuticgoal are also intended to be part of the invention.

In one embodiment, the invention provides a method of treating AS in asubject comprising administering a human TNFα antibody, orantigen-binding portion thereof, e.g., adalimumab, to the subject atweek 0 on a biweekly dosing regimen. In one embodiment, the human TNFαantibody, or antigen-binding portion thereof, is administeredsubcutaneously. In one embodiment, AS is treated by administering ahuman TNFα antibody, or antigen-binding portion thereof, on biweeklydosing regimen for a minimum time period, e.g., at least about 12 weeks,at least about 20, or at least about 24.

Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of active compound calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the active compound and the particulartherapeutic or prophylactic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

Dosage regimens described herein may be adjusted to provide the optimumdesired response, e.g., attaining partial remission of AS, inconsideration of the teachings herein. It is to be noted that dosagevalues may vary with the type and severity of AS. It is to be furtherunderstood that for any particular subject, specific dosage regimens maybe adjusted over time according to the teachings of the specificationand the individual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat dosage amounts and ranges set forth herein are exemplary only andare not intended to limit the scope or practice of the claimedinvention.

Articles of Manufacture

The invention also provides a packaged pharmaceutical compositionwherein the TNFα inhibitor, e.g., TNFα antibody, is packaged within akit or an article of manufacture. The kit or article of manufacture ofthe invention contains materials useful for the treatment, includinginduction and/or remission, prevention and/or diagnosis of AS. The kitor article of manufacture comprises a container and a label or packageinsert or printed material on or associated with the container whichprovides information regarding use of the TNFα inhibitor, e.g., a TNFαantibody, for the treatment of AS.

A kit or an article of manufacture refers to a packaged productcomprising components with which to administer a TNFα inhibitor fortreatment of AS. The kit preferably comprises a box or container thatholds the components of the kit. The box or container is affixed with alabel or a Food and Drug Administration approved label, including aprotocol for administering the TNFα inhibitor. The box or containerholds components of the invention which are preferably contained withinplastic, polyethylene, polypropylene, ethylene, or propylene vessels.The vessels can be capped-tubes or bottles. The kit can also includeinstructions for administering the TNFα antibody of the invention. Inone embodiment the kit of the invention includes the formulationcomprising the human antibody adalimumab (or D2E7), as described inPCT/IB03/04502 and U.S. application Ser. No. 10/222,140, incorporated byreference herein.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

Suitable containers for the TNFα inhibitor, e.g., a TNFα antibody,include, for example, bottles, vials, syringes, pens, etc. Thecontainers may be formed from a variety of materials such as glass orplastic. The container holds a composition which is by itself or whencombined with another composition effective for treating, preventingand/or diagnosing the condition and may have a sterile access port.

In one embodiment, the article of manufacture comprises a TNFαinhibitor, e.g., a TNFα antibody, and a label which indicates to asubject who will be administering the TNFα inhibitor about using theTNFα inhibitor for the treatment of AS. The label may be anywhere withinor on the article of manufacture. In one embodiment, the article ofmanufacture comprises a container, such as a box, which comprises theTNFα inhibitor and a package insert or label providing informationpertaining to use of the TNFα inhibitor for the treatment of AS. Inanother embodiment, the information is printed on a label which is onthe outside of the article of manufacture, in a position which isvisible to prospective purchasers.

In one embodiment, the package insert of the invention informs a reader,including a subject, e.g., a purchaser, who will be administering theTNFα inhibitor for treatment, that the TNFα inhibitor, e.g., a TNFαantibody such as adalimumab, is an indicated treatment of AS, includingof moderately to severely active disease in adult patients.

In one embodiment, the article of manufacture of the invention comprisesa human TNFα antibody, or antigen-binding portion thereof, and a packageinsert comprising instructions for administering the human TNFαantibody, or antigen-binding portion thereof, to a human subject for thetreatment of adults with moderate to severe active ankylosingspondylitis who have had an inadequate response to conventional therapy.

The package insert may describe certain patient populations who mayrespond favorably to the TNFα inhibitor within the article ofmanufacture. For example, the package insert may indicate that the TNFαantibody, e.g., adalimumab, may be used to treat AS in patients who havehad an inadequate response to conventional therapy, e.g., DMARD or NSAIDtherapy. In one embodiment, the invention provides an article ofmanufacture comprising a human TNFα antibody, or antigen-binding portionthereof, and a package insert which indicates that the human TNFαantibody, or antigen-binding portion thereof, is indicated for thetreatment of adults with moderate to severe active ankylosingspondylitis who have had an inadequate response to conventional therapy.

In one embodiment, the package insert of the invention describes certaintherapeutic benefits of the TNFα antibody, e.g., adalimumab, includingspecific symptoms of AS which may be reduced by using the TNFα antibody,e.g., adalimumab. It should be noted that the package insert may alsocontain information pertaining to other disorders which are treatableusing the TNFα antibody, e.g., adalimumab.

In another embodiment, the package insert of the invention describes thedose and administration of adalimumab, for the treatment of AS. Thelabel may indicate that the recommended dose for the treatment of ASwith adalimumab is 40 mg administered every other week. In oneembodiment, the package insert of the invention indicates thatadalimumab is administered by subcutaneous injection.

The package insert of the invention may also provide information tosubjects who will be receiving adalimumab regarding combination uses forboth safety and efficacy purposes. The package insert of the inventionmay contain warnings and precautions regarding the use of the TNFαinhibitor, e.g., a TNFα antibody such as adalimumab.

The label of the invention may further contain information regarding theuse of the TNFα inhibitor, e.g., a TNFα antibody such as adalimumab, inclinical studies for AS. In one embodiment, the label of the inventiondescribes the studies described herein as the Examples, either as awhole or in portion.

In one embodiment of the invention, the kit comprises a TNFα inhibitor,such as an antibody, an second pharmaceutical composition comprising anadditional therapeutic agent, and instructions for administration ofboth agents for the treatment of AS. The instructions may describe how,e.g., subcutaneously, and when, e.g., at week 0, week 2, and biweeklythereafter, doses of TNFα antibody and/or the additional therapeuticagent shall be administered to a subject for treatment.

Another aspect of the invention pertains to kits containing apharmaceutical composition comprising an anti-TNFα antibody and apharmaceutically acceptable carrier and one or more additionalpharmaceutical compositions each comprising a drug useful for treating aTNFα related disorder and a pharmaceutically acceptable carrier.Alternatively, the kit comprises a single pharmaceutical compositioncomprising an anti-TNFα antibody, one or more drugs useful for treatinga TNFα related disorder and a pharmaceutically acceptable carrier. Thekits further contain instructions for dosing of the pharmaceuticalcompositions for the treatment of a TNFα related disorder.

The package or kit alternatively may contain the TNFα inhibitor and itmay be promoted for use, either within the package or throughaccompanying information, for the uses or treatment of the disordersdescribed herein. The packaged pharmaceuticals or kits further caninclude a second agent (as described herein) packaged with or copromotedwith instructions for using the second agent with a first agent (asdescribed herein).

Additional Therapeutic Agents

Methods, uses, and compositions of the invention also includecombinations of TNFα inhibitors, including antibodies, and othertherapeutic agents. TNFα inhibitors, including antibodies, or antigenbinding portions thereof, can be used alone or in combination withadditional agents to treat AS. It should be understood that antibodies,or antigen binding portion thereof, can be used alone or in combinationwith an additional agent, e.g., a therapeutic agent, said additionalagent being selected by the skilled artisan for its intended purpose.For example, the additional agent can be a therapeutic agentart-recognized as being useful to treat the disease or condition beingtreated by the antibody of the present invention. The additional agentalso can be an agent that imparts a beneficial attribute to thetherapeutic composition e.g., an agent which affects the viscosity ofthe composition.

It should further be understood that the combinations which are to beincluded within this invention are those combinations useful for theirintended purpose. The agents set forth below are illustrative forpurposes and not intended to be limited. The combinations, which arepart of this invention, can be the antibodies of the present inventionand at least one additional agent selected from the lists below. Thecombination can also include more than one additional agent, e.g., twoor three additional agents if the combination is such that the formedcomposition can perform its intended function.

TNFα inhibitors described herein may be used in combination withadditional therapeutic agents such as a Disease Modifying Anti-RheumaticDrug (DMARD) or a Nonsteroidal Antiinflammatory Drug (NSAID) or asteroid or any combination thereof. Preferred examples of a DMARD arehydroxychloroquine, leflunomide, methotrexate, parenteral gold, oralgold and sulfasalazine. Preferred examples of non-steroidalanti-inflammatory drug(s) also referred to as NSAIDS include drugs likeibuprofen. Other preferred combinations are corticosteroids includingprednisolone; the well known side effects of steroid use can be reducedor even eliminated by tapering the steroid dose required when treatingpatients in combination with the anti-TNFα antibodies of this invention.Non-limiting examples of therapeutic agents for rheumatoid arthritiswith which an antibody, or antibody portion, of the invention can becombined include the following: cytokine suppressive anti-inflammatorydrug(s) (CSAIDs); antibodies to or antagonists of other human cytokinesor growth factors, for example, TNF, LT, IL-1, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, IL-21, IL-23, interferons,EMAP-II, GM-CSF, FGF, and PDGF. Antibodies of the invention, or antigenbinding portions thereof, can be combined with antibodies to cellsurface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40,CD45, CD69, CD80 (B7.1), CD86 (B7.2), CD90, CTLA or their ligandsincluding CD154 (gp39 or CD40L).

Preferred combinations of therapeutic agents may interfere at differentpoints in the autoimmune and subsequent inflammatory cascade; preferredexamples include TNF antagonists/inhibitors such as soluble p55 or p75TNF receptors, derivatives, thereof, (p75TNFR1gG (Enbrel™) or p55TNFR1gG(Lenercept), chimeric, humanized or human TNF antibodies, or a fragmentthereof, including infliximab (Remicade®, Johnson and Johnson; describedin U.S. Pat. No. 5,656,272, incorporated by reference herein), CDP571 (ahumanized monoclonal anti-TNF-alpha IgG4 antibody), CDP 870 (a humanizedmonoclonal anti-TNF-alpha antibody fragment), an anti-TNF dAb (Peptech),CNTO 148 (golimumab; Medarex and Centocor, see WO 02/12502), andadalimumab (Humira® Abbott Laboratories, a human anti-TNF mAb, describedin U.S. Pat. No. 6,090,382 as D2E7). Additional TNF antibodies which canbe used in the invention are described in U.S. Pat. Nos. 6,593,458;6,498,237; 6,451,983; and 6,448,380, each of which is incorporated byreference herein. Other combinations including TNFα converting enzyme(TACE) inhibitors; IL-1 inhibitors (Interleukin-1-converting enzymeinhibitors, IL-1RA etc.) may be effective for the same reason. Othercombinations include the IL-6 antibody tocilizumab (Actemra). Otherpreferred combinations include Interleukin 11. Yet another preferredcombination are other key players of the autoimmune response which mayact parallel to, dependent on or in concert with TNFα function;especially preferred are IL-18 antagonists including IL-18 antibodies orsoluble IL-18 receptors, or IL-18 binding proteins. It has been shownthat TNFα and IL-18 have overlapping but distinct functions and acombination of antagonists to both may be most effective. Yet anotherpreferred combination are non-depleting anti-CD4 inhibitors. Yet otherpreferred combinations include antagonists of the co-stimulatory pathwayCD80 (B7.1) or CD86 (B7.2) including antibodies, soluble receptors orantagonistic ligands.

The TNFα inhibitors, including antibodies, or antigen binding portionsthereof, used in the invention may also be combined with agents, such asmethotrexate, 6-MP, azathioprine sulphasalazine, mesalazine, olsalazinechloroquinine/hydroxychloroquine, pencillamine, aurothiomalate(intramuscular and oral), azathioprine, cochicine, corticosteroids(oral, inhaled and local injection), beta-2 adrenoreceptor agonists(salbutamol, terbutaline, salmeteral), xanthines (theophylline,aminophylline), cromoglycate, nedocromil, ketotifen, ipratropium andoxitropium, cyclosporin, FK506, rapamycin, mycophenolate mofetil,leflunomide, NSAIDs, for example, ibuprofen, corticosteroids such asprednisolone, phosphodiesterase inhibitors, adensosine agonists,antithrombotic agents, complement inhibitors, adrenergic agents, agentswhich interfere with signaling by proinflammatory cytokines such as TNFαor IL-1 (e.g. IRAK, NIK, IKK, p38 or MAP kinase inhibitors), IL-1βconverting enzyme inhibitors, TNFα converting enzyme (TACE) inhibitors,T-cell signalling inhibitors such as kinase inhibitors,metalloproteinase inhibitors, sulfasalazine, azathioprine,6-mercaptopurines, angiotensin converting enzyme inhibitors, solublecytokine receptors and derivatives thereof (e.g. soluble p55 or p75 TNFreceptors and the derivatives p75TNFRIgG (Enbrel™ and p55TNFRIgG(Lenercept)), sIL-1 RI, sIL-IRII, sIL-6R), antiinflammatory cytokines(e.g. IL-4, IL-10, IL-11, IL-13 and TGFβ), tocilizumab (Actemra),celecoxib, folic acid, hydroxychloroquine sulfate, rofecoxib,etanercept, infliximab, naproxen, valdecoxib, sulfasalazine,methylprednisolone, meloxicam, methylprednisolone acetate, gold sodiumthiomalate, aspirin, triamcinolone acetonide, propoxyphenenapsylate/apap, folate, nabumetone, diclofenac, piroxicam, etodolac,diclofenac sodium, oxaprozin, oxycodone hcl, hydrocodonebitartrate/apap, diclofenac sodium/misoprostol, fentanyl, anakinra,human recombinant, tramadol hcl, salsalate, sulindac,cyanocobalamin/fa/pyridoxine, acetaminophen, alendronate sodium,prednisolone, morphine sulfate, lidocaine hydrochloride, indomethacin,glucosamine sulf/chondroitin, amitriptyline hcl, sulfadiazine, oxycodonehcl/acetaminophen, olopatadine hcl, misoprostol, naproxen sodium,omeprazole, cyclophosphamide, rituximab, IL-1 TRAP, MRA, CTLA4-IG, IL-18BP, anti-IL-18, Anti-IL15, BIRB-796, SCIO-469, VX-702, AMG-548, VX-740,Roflumilast, IC-485, CDC-801, and Mesopram. Preferred combinationsinclude methotrexate or leflunomide and in moderate or severe AS cases,cyclosporine.

Non-limiting examples of therapeutic agents for AS with which TNFαinhibitor, such as an antibody, or antibody portion, can be combinedinclude the following: methotrexate, etanercept, rofecoxib, celecoxib,folic acid, sulfasalazine, naproxen, leflunomide, methylprednisoloneacetate, indomethacin, hydroxychloroquine sulfate, prednisone, sulindac,betamethasone diprop augmented, infliximab, methotrexate, folate,triamcinolone acetonide, diclofenac, dimethylsulfoxide, piroxicam,diclofenac sodium, ketoprofen, meloxicam, methylprednisolone,nabumetone, tolmetin sodium, calcipotriene, cyclosporine, diclofenacsodium/misoprostol, fluocinonide, glucosamine sulfate, gold sodiumthiomalate, hydrocodone bitartrate/apap, ibuprofen, risedronate sodium,sulfadiazine, thioguanine, valdecoxib, alefacept, efalizumab.

Non-limiting examples of therapeutic agents for Ankylosing Spondylitiswith which an antibody, or antibody portion, of the invention can becombined include the following: ibuprofen, diclofenac and misoprostol,naproxen, meloxicam, indomethacin, diclofenac, celecoxib, rofecoxib,Sulfasalazine, Methotrexate, azathioprine, minocyclin, prednisone,etanercept, infliximab. In one embodiment, the methods of the inventioninclude the combination of a TNFα inhibitor and methotrexate.

IV. Efficacy of TNFα Inhibitor

The invention also provides methods for determining whether a TNFαinhibitor is effective at treating AS in a subject. Such methods may beused to determine the efficacy of a TNFα inhibitor, including thosewhich are unknown or unconfirmed to have such efficacy. Using themethods described herein, effective TNFα inhibitors may be determined orconfirmed, and, subsequently, used in the method of treating AS.

Methods of determining efficacy of a TNFα inhibitor for treatingankylosing spondylitis (AS) in a subject include any means known in theart. The clinical course of AS is measured by using any number ofinstruments to evaluate various AS symptoms. Some of the commonly usedscales include the Assessment in Ankylosing Spondylitis (ASAS), the BathAnkylosing Spondylitis Disease Activity Index (BASDAI) (Garrett et al.(1994) J Rheumatol 21:2286), the Bath Ankylosing Spondylitis MetrologyIndex (BASMI) (Jenkinson et al. (1994) J Rheumatol 21:1694), and theBath Ankylosing Spondylitis Functional Index (BASFI) (Calin et al.(1994) J Rheumatol 21:2281). These indices can be used to monitor apatient over time and to determine improvement. Each of these scales isdescribed further below, as well as in the examples:

Criteria for Measuring the Clinical Course of AS

1. The Assessment in Ankylosing Spondylitis (ASAS20) is the primaryendpoint in the pivotal Phase 3 AS studies. A ≧20% improvement andabsolute improvement of ≧210 units (scale of 0-100) in ≧3 of 4 domains:Subject Global Assessment, Pain, Function, and Inflammation. There mustbe an absence of deterioration in the potential remaining domain(deterioration is defined as a change for the worse of ≧20% and a networsening of ≧10 units (scale of 0-100).2. The Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) canbe used to evaluate the level of disease activity in a patient with AS.BASDAI focuses upon signs and symptoms of the inflammatory aspects ofAS, nocturnal and total back pain, the patient's global assessment andactual physical measurements of spinal mobility such as the Schober'stest, chest expansion score and occiput to wall measurement. BASDAImeasures disease activity on the basis of six questions relating tofatigue, spinal pain, peripheral arthritis, enthesitis (inflammation atthe points where tendons/ligaments/joint capsule enter the bone), andmorning stiffness. These questions are answered on a 10-cm horizontalvisual analog scale measuring severity of fatigue, spinal and peripheraljoint pain, localized tenderness, and morning stiffness (bothqualitative and quantitative). The final BASDAI score has a range of 0to 10.3. The Bath Ankylosing Spondylitis Functional Index (BASFI) measures thephysical function impairment caused by AS, and is a self-assessmentinstrument that consists of 8 specific questions regarding function inAS, and 2 questions reflecting the patient's ability to cope witheveryday life. Each question is answered on a 10-cm horizontal visualanalog scale, the mean of which gives the BASFI score (0-10).4. The Bath Ankylosing Spondylitis Metrology Index (BASMI) consists of 5simple clinical measurements that provide a composite index and definedisease status in AS. Analysis of metrology (20 measurements) identifiedthese 5 measurements as most accurately reflecting axial status:cervical rotation, tragus to wall distance, lateral flexion, modifiedSchober's test, and internalleolar distance. The BASMI is quick (7minutes), reproducible, and sensitive to change across the entirespectrum of disease. The BASMI index comprises 5 measures of hip andspinal mobility in AS. The five BASMI measures, scaled 0 (mild) to 10(severe), include tragus to wall distance, cervical rotation, lumbarflexion, lumbar side flexion, and intermolleolar distance.

Combinations of the above-mentioned criteria may also used to evaluatepatients. In addition, other indices such as FACIT-F or the ability ofthe TNFα inhibitor to induce partial remission can be used to determinedisease activity in AS patients.

The invention provides methods for determining whether a TNFα inhibitoris effective at treating AS in a subject. Such methods may be used todetermine the efficacy of a TNFα inhibitor, including those which areunknown or unconfirmed to have such efficacy. Using the methodsdescribed herein, effective TNFα inhibitors may be determined orconfirmed, and, subsequently, used in the method of treating AS.

In one embodiment, the invention provides a method for determiningefficacy using a Bath Ankylosing Spondylitis Disease Activity Index(BASDAI) 20 response. The invention includes a method of determining theefficacy of a TNFα inhibitor for treating ankylosing spondylitis (AS) ina subject comprising determining a Bath Ankylosing Spondylitis DiseaseActivity Index (BASDAI) 20 response of a patient population having ASand who was administered the TNFα inhibitor, wherein an BASDAI 20response in at least about 60% of the patient population indicates thatthe TNFα inhibitor is an effective TNFα inhibitor for the treatment ofAS in a subject.

The invention further provides a method of treating AS in a subjectcomprising administering an effective TNFα inhibitor to the subject suchthat AS is treated, wherein the effective TNFα inhibitor was previouslyidentified as resulting in a BASDAI 20 response in at least about 60% ofa patient population having AS and who was administered the TNFαinhibitor. In one embodiment, the effective TNFα inhibitor waspreviously identified as resulting in a BASDAI 20 response in at leastabout 65% of a patient population having AS and who was administered theTNFα inhibitor. In one embodiment, the effective TNFα inhibitor waspreviously identified as resulting in a BASDAI 20 response in at leastabout 70% of a patient population having AS and who was administered theTNFα inhibitor. In one embodiment, the effective TNFα inhibitor waspreviously identified as resulting in a BASDAI 20 response in at leastabout 75% of a patient population having AS and who was administered theTNFα inhibitor. In one embodiment, the effective TNFα inhibitor waspreviously identified as resulting in a BASDAI 20 response in at leastabout 80% of a patient population having AS and who was administered theTNFα inhibitor. In one embodiment, the effective TNFα inhibitor waspreviously identified as resulting in a BASDAI 20 response in at leastabout 85% of a patient population having AS and who was administered theTNFα inhibitor. In one embodiment, an BASDAI 20 response in at leastabout 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, of thepatient population indicates that the TNFα inhibitor is an effective TNFinhibitor for the treatment of AS in the subject.

The invention also includes a method of determining the efficacy of aTNFα inhibitor for treating ankylosing spondylitis (AS) in a subjectcomprising determining a Bath Ankylosing Spondylitis Disease ActivityIndex (BASDAI) 50 response of a patient population having AS and who wasadministered the TNFα inhibitor, wherein an BASDAI 50 response in atleast about 65% of the patient population indicates that the TNFαinhibitor is an effective TNFα inhibitor for the treatment of AS in thesubject. In one embodiment, an BASDAI 50 response in at least about 23%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, or at least about60%, of the patient population indicates that the TNFα inhibitor is aneffective TNF inhibitor for the treatment of AS in the subject. In oneembodiment, an BASDAI 50 response in at least about 23%, 24%, 25%, 26%,27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%. 35%, 36%, 37%, 38%, 39%, 40%,41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60% of the patient population indicates thatthe TNFα inhibitor is an effective TNF inhibitor for the treatment of ASin the subject.

The invention further provides a method of determining the efficacy of aTNFα inhibitor for treating ankylosing spondylitis (AS) in a subjectcomprising determining a Bath Ankylosing Spondylitis Disease ActivityIndex (BASDAI) 70 response of a patient population having AS and who wasadministered the TNFα inhibitor, wherein an BASDAI 70 response in atleast about 10% of the patient population indicates that the TNFαinhibitor is an effective TNFα inhibitor for the treatment of AS in thesubject. In one embodiment, an BASDAI 50 response in at least about 15%,at least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, of the patientpopulation indicates that the TNFα inhibitor is an effective TNFinhibitor for the treatment of AS in the subject. In one embodiment, anBASDAI 50 response in at least about 23%, 24%, 25%, 26%, 27%, 28%, 29%,30%, 31%, 32%, 33%, 34%. 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%,44%, 45%, of the patient population indicates that the TNFα inhibitor isan effective TNF inhibitor for the treatment of AS in the subject.

The invention provides a method of treating AS in a subject comprisingadministering an effective TNFα inhibitor to the subject such that AS istreated, wherein the effective TNFα inhibitor was previously identifiedas resulting in an ASAS20 response in at least about 61% of a patientpopulation having AS who was administered the TNFα inhibitor. In oneembodiment, an ASAS20 response in at least about 27%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 55%, at least about 60%, at least about65%, or at least about 70% of the patient population indicates that theTNFα inhibitor is an effective TNFα inhibitor for the treatment of AS inthe subject. In one embodiment, an ASAS20 response in at least about27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%. 35%, 36%, 37%, 38%, 39%, 40%,41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, or 73% of the patient population indicates that theTNFα inhibitor is an effective TNFα inhibitor for the treatment of AS inthe subject

The invention also includes a method of determining the efficacy of aTNFα inhibitor for treating ankylosing spondylitis (AS) in a subjectcomprising determining a Assessment in Apkylosing Spondylitis (ASAS)response of a patient population having AS and who was administered theTNFα inhibitor, wherein an ASAS40 response in at least about 39% of thepatient population indicates that the TNFα inhibitor is an effectiveTNFα inhibitor for the treatment of AS in the subject. In oneembodiment, an ASAS40 response in at least about 40% of the patientpopulation indicates that the TNFα inhibitor is an effective TNFαinhibitor for the treatment of AS in the subject. In one embodiment, anASAS40 response in at least about 41% of the patient populationindicates that the TNFα inhibitor is an effective TNFα inhibitor for thetreatment of AS in the subject.

The invention also includes a method of determining the efficacy of aTNFα inhibitor for treating ankylosing spondylitis (AS) in a subjectcomprising determining a Assessment in Ankylosing Spondylitis (ASAS)response of a patient population having AS and who was administered theTNFα inhibitor, wherein an ASAS50 response in at least about 39% of thepatient population indicates that the TNFα inhibitor is an effectiveTNFα inhibitor for the treatment of AS in the subject. In oneembodiment, an ASAS50 response in at least about 40% of the patientpopulation indicates that the TNFα inhibitor is an effective TNFαinhibitor for the treatment of AS in the subject.

The invention also includes a method of determining the efficacy of ahuman TNFα antibody or for treating ankylosing spondylitis (AS) in asubject comprising determining a Assessment in Ankylosing Spondylitis(ASAS) 70 response of a patient population having AS and who wasadministered the human TNFα antibody, or antigen-binding portionthereof, wherein an ASAS70 response in at least about 5% of the patientpopulation indicates that the human TNFα antibody, or antigen-bindingportion thereof, is an effective human TNFα antibody, or antigen-bindingportion thereof, for the treatment of AS in the subject. In oneembodiment, an ASAS70 response in at least about 20% of the patientpopulation indicates that the human TNFα antibody, or antigen-bindingportion thereof, is an effective human TNFα antibody, or antigen-bindingportion thereof, for the treatment of AS in the subject. In oneembodiment, an ASAS70 response in at least about 25% of the patientpopulation indicates that the human TNFα antibody, or antigen-bindingportion thereof, is an effective human TNFα antibody, or antigen-bindingportion thereof, for the treatment of AS in the subject. In oneembodiment, an ASAS70 response in at least about 30% of the patientpopulation indicates that the human TNFα antibody, or antigen-bindingportion thereof, is an effective human TNFα antibody, or antigen-bindingportion thereof, for the treatment of AS in the subject. In oneembodiment, an ASAS70 response in at least about 40% of the patientpopulation indicates that the human TNFα antibody, or antigen-bindingportion thereof, is an effective human TNFα antibody, or antigen-bindingportion thereof, for the treatment of AS in the subject. In oneembodiment, an ASAS70 response in at least about 5%, at least about 20%,at least about 24%, or at least about 40% of the patient populationindicates that the TNFα inhibitor is an effective TNFα inhibitor for thetreatment of AS in the subject. In one embodiment, an ASAS70 response inat least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%,17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%,31%, 32%, 33%, 34%. 35%, 36%, 37%, 38%, 39%, or 40% of the patientpopulation indicates that the TNFα inhibitor is an effective TNFαinhibitor for the treatment of AS in the subject.

Numbers intermediate to any percentages recited herein, including thosein the Examples, e.g., 61%, 62%, 63%. 64%, 65%, 66%, 67%, 68%, 69%, arealso intended to be part of this invention. Ranges of values using acombination of any of the above recited values as upper and/or lowerlimits are intended to be included in the scope of the invention

The invention may also include further comprising administering theeffective human TNFα antibody, or antigen-binding portion thereof,described herein to a subject to treat AS.

Also encompassed in the invention is a method of treatment comprisingadministering a TNFα inhibitor shown to be efficacious according to themethods described herein and in the Examples. In one embodiment, themethods of the invention comprise administering the TNFα inhibitor tothe subjects of a patient population and determining the efficacy of theTNFα inhibitor by determining changes, improvements, measurements, etc.,using AS indices known in the art, in the patient population incomparison to the Examples set forth below. For example, the inventionfurther provides a method of treating AS in a subject comprisingadministering an effective TNFα inhibitor to the subject such that AS istreated, wherein the effective TNFα inhibitor was previously identifiedas resulting in a BASDAI 50 response in at least about 60% of a patientpopulation having AS and who was administered the TNFα inhibitor.

It should be noted that the Examples provided herein represent differentmethods of determining the efficacy of a TNFα inhibitor, such as a humanTNFα antibody, or antigen-binding portion thereof. As such, data andresults described in the Examples section which shows efficacy of a TNFαinhibitor, e.g., ability to treat AS, are included in the methods ofdetermining efficacy of the invention.

Time points for determining efficacy will be understood by those ofskill in the art to depend on the type of efficacy being determined,e.g., induction of partial remission. In one embodiment, measurements inscores, e.g., an improvement in the ASAS20 response, may be measuredagainst a subject's baseline score. Generally, a baseline refers to ameasurement or score of a patient before treatment, i.e. week 0. Othertime points may also be included as a starting point in determiningefficacy, however.

Patient populations described in the methods of the invention aregenerally selected based on common characteristics, such as, but notlimited to, subjects diagnosed with AS. Such a patient population wouldbe appropriate, for example, for determining the efficacy of the TNFαinhibitor for inducing partial remission in AS in the given patientpopulation. In one embodiment, the patient population is an adultpopulation.

In one embodiment, the methods of the invention for determining whethera TNFα inhibitor is an effective TNFα inhibitor, include determiningchanges, improvements, measurements, etc., in AS using appropriateindices described herein, e.g., ASAS responses, BASDAI, from a patientpopulation who has already been administered the TNFα inhibitor. Such apatient population may be pre-selected according to commoncharacteristics, e.g., AS, and may have already been given the TNFαinhibitor. Administration of the TNFα inhibitor may or may not beperformed by the same person of ordinary skill who is determining theefficacy of the TNFα inhibitor in accordance with the teachings of thespecification.

Methods of the invention relating to determining efficacy, i.e.,determining whether a TNFα inhibitor is an effective TNFα inhibitor, mayalso be applied to specific patient populations within the overallpatient population who together have specific, common characteristics,i.e., a subpopulation. In addition, while the above methods aredescribed in terms of patient populations, methods of efficacy describedherein may also be applied to individual subjects.

The Examples and discoveries described herein are representative of aTNFα inhibitor, i.e., adalimumab, which is effective for treating AS,including reducing pain and fatigue and inducing partial remission ofAS. As such, the studies and results described in the Examples sectionherein may be used as a guideline for determining the efficacy of a TNFαinhibitor, i.e., whether a TNFα inhibitor is an effective TNFα inhibitorfor the treatment of AS. In one embodiment, methods of determiningefficacy described herein may be used to determine whether a TNFαinhibitor is bioequivalent to another TNFα inhibitor.

The present invention is further illustrated by the following exampleswhich should not be construed as limiting in any way.

EXAMPLES Example 1 Adalimumab in the Treatment of Active AnkylosingSpondylitis: Results of an Open-Label, 52-Week Trial

Tumor necrosis factor (TNF) antagonists infliximab and etanercept haveshown efficacy in the treatment of ankylosing spondylitis (AS).Adalimumab (Abbott Laboratories) is a fully human, anti-TNF monoclonalantibody that reduces the signs and symptoms and progression of diseaseof rheumatoid arthritis and has been evaluated in AS over 20 weeks.

The objective of this study was to examine the potential therapeuticeffects of adalimumab in NSAID-refractory AS patients who were treatedfor 52 weeks (Ann Rheum Dis 2005; 64(Suppl III):316). To further thisobjective, fifteen patients were enrolled (patient characteristics aredetailed in Table 1). All patients suffered from spinal pain, and 4patients also had peripheral arthritis. Adalimumab 40 mg wasadministered subcutaneously every other week (eow). Clinical outcomeassessments included disease activity (BASDAI), function (BASFI),metrology (BASMI), patient's and physician's global and nocturnalassessments of pain (NRS), peripheral joint assessment, Maastrichtenthesitis score, quality of life (SF-36), and C-reactive protein (CRP).The primary endpoint of this study was improvement of disease activity(BASDAI 50%) at Week 12. Outcome parameters are listed in Table 2.

TABLE 1 Patient Characteristics. Number of patients (n) 15 Male/female(n) 9/6 Mean age, years (range) 40 (19-55) Mean disease duration, years(range) 11 (2-33) HLA-B27 positive 86% Mean BASDAI (range) 6.6 (4.7-8.5)Patients with joint involvement (n)  4

TABLE 2 Outcome Parameters. Baseline Week 2 Week 12 Week 52 BASFI 6.35.1 4.3 3.7 BASMI 4.5 3.8 3.9 3.4 General Pain 7.4 5.1 4.1 3.4 NocturnalPain 7.3 4.7 4.0 3.2 Patient's global 7.3 5.0 4.3 3.1

Thirteen patients completed the 52-week therapy. One patient withdrewafter 8 weeks for personal reasons; and the other patient withdrewbecause of inefficacy and remitting minor infections at Week 28.

At Week 52, the BASDAI showed a significant improvement (see FIG. 1).For example, at 52 weeks, over 75% of patients treated with adalimumabhad achieved a BASDAI20 response, about 60% had achieved a BASDAI50response, and more than 45% had achieved a BASDAI70 response.

Similar levels of improvement were achieved in applying the Assessmentof Ankylosing Spondylitis (ASAS) working group improvement criteria (seeFIG. 2). At 52 weeks, about 73% of patients treated with adalimumab hadachieved an ASAS20 response, about 70% achieved an ASAS40 response, andabout 40% achieved an ASAS70 response.

In addition, the CRP, BASFI, patient's and physician's globalassessments, general and nocturnal assessments of pain (NRS), BASMI,morning stiffness (BASDAI Question 4 and 5) and the Physical ComponentSummary (PCS) of SF-36 improved significantly. Adalimumab waswell-tolerated, and no serious infections occurred during the study. Inthis open-label study, adalimumab showed significant and sustainedimprovement of spinal symptoms in active AS over 1 year.

Example 2 Adalimumab Reduces Fatigue in Patients with Active AnkylosingSpondylitis (AS)

Fatigue, defined as enduring, subjective sensation of generalizedtiredness or exhaustion, has been increasingly recognized as animportant outcome measure in AS (Dagfinrud et al. Arth Rheum 2005;53(1):5-11; Jones S D et al. J Rheumatol 1996; 23(3):487-90; Haywood H Let al. Rheumatol 2002; 41:1295-1302; Ward M M. Arth Care Res 1999;12:247-55; Van Tubergen et al. Arth Rheum 2002; 27(1):8-16). It has beenreported that 65% of people living with AS describe fatigue as a majorsymptom from time to time (Jones S D et al. J Rheumatol 1996;23(3):487-90). The objective of this study was to evaluate the impact ofadalimumab therapy (a TNF antagonist) on fatigue in active AS patients.

Overview

This phase III, double-blind, randomized, placebo-controlled trial wasconducted at 11 sites in Canada (study design is shown in FIG. 3). Thestudy enrolled active AS patients with an inadequate response to atleast one NSAID. The study included a 24-week study period of 40 mgadalimumab subcutaneous (sc) injection every other week (eow) orplacebo.

There was an early escape option to open label 40 mg adalimumab sc eowat Weeks 12, 16, or 20. Patients completed a questionnaire on diseaseactivity (BASDAI [Bath Ankylosing Spondylitis Disease Activity Index]).Fatigue was assessed by patient self-reported questionnaires, includingFACIT-Fatigue, BASDAI Fatigue item, and SF-36 Vitality domain.

Instruments for Fatigue Assessments

FACIT-Fatigue is a widely used measure of fatigue in chronic illnesses.This measure contains 13 items pertaining to the past 7 days to be ratedon a 5-point Likert scale. Scores range from 0-52, with higher scoresrepresenting less fatigue. A 3-point or more change is consideredclinically meaningful (Cella et al. Cancer 2002; 94:528-538; and Cellaet al. J Clin Oncol 2003; 21:366). FACIT-Fatigue was administered atBaseline and at Weeks 2, 12, and 24.

The BASDAI scale is widely used in clinical studies to evaluate ASdisease activity. The BASDAI is a six-item measure of disease activityand includes questions on fatigue, spinal pain, peripheral arthritis,enthesitis, and duration and severity of morning stiffness. The BASDAIscale has six items pertaining to the past 7 days on a 10-cm visualanalog scale (VAS). Fatigue is the first item on the BASDAI scale,asking the subject to rate “overall degree of tiredness” during the past7 days on a 10-cm VAS. BASDAI Fatigue Item scores range from 0-10, withlower scores representing less fatigue. BASDAI was administered atBaseline and at Weeks 2, 4, 8, 12, 16, 20, and 24

SF-36 is a widely applied instrument for measuring health status, andconsists of 8 domains: physical function, bodily pain, rolelimitations-physical, general health, vitality, social function, rolelimitations-emotional, and mental health. The Vitality domain has fouritems to measure energy level and fatigue. SF-36 has a 4-week recallperiod, and domain scores range from 0-100, with higher scoresreflecting better health status. A 5-10 point change in domain scores isconsidered clinically meaningful (Kosinski M et al. Arth Rheum 2000;7:1478-1487). SF-36 also contains 2 summary scores, the mental componentsummary (MCS) and the physical component summary (PCS); a 2.5-5 pointchange in summary scores is considered clinically meaningful (Kosinski Met al. Arth Rheum 2000; 7:1478-1487). SF-36 was administered at Baselineand at Weeks 12 and 24.

Patient disposition at Week 24 is shown in Table 3. Baselinedemographics and disease activity are shown in Table 4. Patient-ReportedFatigue and Health-Related Quality of Life (HRQL) Scores at Baseline areshown in Table 5.

TABLE 3 Patient Disposition at Week 24 Adalimumab Placebo 40 mg eow (N =44) (N = 38) n (%) n (%) Subjects Randomized 44 38 Subjects Treated 4438 Subjects Completing Week 24 44 (100)  38 (100) Prematurely terminatedat 2 (4.5) 0 Week 24 AE  0 0 Withdrew consent 1 (2.3) 0 Lost tofollow-up  0 0 Other 1 (2.3) 0

TABLE 4 Baseline Demographics and Disease Activity Adalimumab Placebo 40mg eow (N = 107) (N = 208) n (%) n (%) p-value Age (years) 43.4 41.70.220a Sex (males) 79 (73.8) 157 (75.5) 0.784b Race (Whites) 99 (92.5)202 (97.1) 0.050, b Weight (kg) 79.8 81.9 0.320 HLA-B27 positive 85(79.4) 163 (78.4) 0.960b Duration of AS 10.0 11.3 0.261a (years) BASDAIscore  6.3  6.3 0.633a

TABLE 5 Patient-Reported Fatigue and Health-Related Quality of Life(HRQL) Score at Baseline* Placebo Adalimumab 40 mg (N = 44) eow (N = 38)p-value FACIT-Fatigue 23.6 (+9.9) 24.4 (+10.8) 0.770 BASDAI Fatigue  6.9(+1.9) 6.3 (+2.3) 0.357 Item SF-36 Vitality  34.6 (+18.4) 30.4 (+17.9)0.325 Domain SF-36 PCS 32.8 (+7.3) 32.9 (+8.3)  0.868 SF-36 MCS  41.6(+10.0) 42.8 (+9.5)  0.517 *Mean + standard deviation

Data from the study is shown below. Patients with active AS experiencedfatigue symptom and impairment in physical functioning at Baseline.Compared with placebo, adalimumab treatment demonstrated statisticallysignificant improvement in disease activity (as measured by BASDAI, seeTable 6)).

TABLE 6 Change from Baseline to Weeks 12 and 24 in DiseaseActivity-BASDAI Score Mean change in BASDAI score* Placebo AdalimumabWeek 12 −0.51 −2.04** Week 24 −0.37 −1.99** *Mean change from baseline(LOCF). **Statistically significant at p ≦ 0.01 level, p-value fordifference between therapies from ANCOVA.

After 12 and 24 weeks of adalimumab therapy, patients reportedstatistically significant and clinically meaningful improvements infatigue symptom and functioning compared with placebo (FACIT-Fatiguescores for the two treatment groups are shown in Table 7 and FIG. 4).

TABLE 7 Change from Baseline to Weeks 12 and 24 in FACIT-Fatigue ScoreMean change in FACIT-Fatigue score^(†) Placebo Adalimumab Week 12 1.77.0** Week 24 2.5 7.8* ^(†)Mean change from baseline (LOCF). *,**Statistically significant at p ≦ 0.05 and p ≦ 0.01 levels,respectively. Minimum Important Difference = 3 Cella et al. Cancer 2002;94: 528-538 Cella et al. J Clin Oncol 2003; 21: 366-373.

A statistically significant difference in BASDAI Fatigue Item Score wasseen between the placebo and adalimumab treatment groups at Weeks 12 and24, as shown in Table 8 and FIG. 5.

TABLE 8 Change from Baseline to Weeks 12 and 24 in BASDAI Fatigue ItemScore Mean change in BASDAI-Fatigue score^(†) Placebo Adalimumab Week 12−0.5 −1.7** Week 24 −0.5 −1.9** ^(†)Mean change from baseline (LOCF).**Statistically significant at p ≦ 0.01 level.

Statistically significant improvements were also seen in the adalimumabtreatment group compared with placebo in the SF-36 Vitality Domain Score(Table 9 and FIG. 6), the SF-36 PCS Score (Table 10), and the SF-36 MCSScore (Table 11).

TABLE 9 Change from Baseline to Weeks 12 and 24 in SF-36 Vitality DomainScore Mean change in SF-36 Vitality Domain Score^(†) Placebo AdalimumabWeek 12 1.3 17 Week 24 2.8 18.1 ^(†)Mean change from baseline (LOCF).Minimum Important Difference = 10 Kosinski M et al. Arth Rheum 2000; 7:1478-1487.

TABLE 10 Change from Baseline in Weeks 12 and 24 in SF-36 PCS Score Meanchange in SF-36 PCS Score^(†) Placebo Adalimumab Week 12 0.9 7.8*** Week24 1.1 6.1*** ^(†)Mean change from baseline (LOCF). ***Statisticallysignificant at p ≦ 0.001 level. Minimum Important Difference = 3Kosinski M et al. Arth Rheum 2000; 7: 1478-1487.

TABLE 11 Change from Baseline in Weeks 12 and 24 in SF-36 MCS ScoreSF-36 MCS Score^(†) Placebo Adalimumab Week 12 0.0 5.4 Week 24 1.1 6.1^(†)Mean change from baseline (LOCF). Minimum Important Difference = 3Kosinski M et al. Arth Rheum 2000; 7: 1478-1487.

In conclusion, these results show that adalimumab treatment may reducefatigue and improve functioning in AS patients.

Example 3 Major Clinical Response and Partial Remission in AnkylosingSpondylitis Subjects Treated with Adalimumab: Study H

Ankylosing Spondylitis (AS) is a common inflammatory rheumatic diseasethat produces progressive spinal stiffness and restriction of mobility.Tumor necrosis factor (TNF) is thought to play a major role in thepathogenesis of AS. No trial of a disease-modifying antirheumatic drug(DMARD) has yielded consistent positive results for the treatment of AS.

Adalimumab is a fully human monoclonal antibody targeting TNF, currentlyapproved for the treatment of rheumatoid arthritis and psoriaticarthritis in the US and Europe, and currently pending approval from theFDA and EMEA for AS. The objective of the study described herein was toinvestigate the ability of adalimumab to effect a major clinicalresponse and partial remission in subjects with ankylosing spondylitis.Partial remission is defined as a value of <20 on a 0-100 VAS scale ineach of the 4 ASAS domains: Pain (Total Back Pain [TBP]), Function (BathAS Functional Index [BASFI], Patient's Global Assessment (PGA) ofdisease activity, and Inflammation (Bath AS Disease Activity Index[BASDAI] questions 5 and 6).

Study H was a phase III, randomized, placebo-controlled, double-blind,multi-center study designed to assess the efficacy and safety ofadalimumab in the treatment of active AS in subjects who had aninadequate response, or were intolerant to, treatment with at least onenonsteroidal anti-inflammatory drug (NSAID). Patients may have also hadinadequate response to at least one DMARD. Thus, the study includedsubjects who had failed previous conventional therapy for AS.

Study H was a 2-year study in which subjects were randomized in a 2:1ratio to receive either 40 mg subcutaneous (sc) doses of adalimumab orplacebo every other week (eow). After the initial 24-week blindedperiod, patients had the option to participate in a subsequent 80-weekopen label extension. The study design of Study H is outlined in FIG. 7.Subjects who failed to achieve an Assessment in Ankylosing Spondylitis(ASAS) 20 response at or after Week 12 were eligible for open-labelearly escape therapy (EET) with adalimumab 40 mg sc eow. Subjectsreceiving early escape therapy were treated as nonresponders at allsubsequent visits. All patients were assessed at Weeks 2, 4, 8, 12, 16,20, and 24.

Patient inclusion criteria were inadequate response to at least oneNSAID, and active AS, defined by fulfillment of at least 2 of thefollowing 3 criteria: Bath AS Disease Activity Index (BASDAI) score ≧4,Visual Analog Scale (VAS) score for Total Back Pain (TBP)≧4, and morningstiffness ≧1 hour. Primary outcome measures were assessed at Weeks 12and 24. Primary outcome measures include ASAS Partial Remission Criteria(value of <20 on a 0-100 scale in each of the 4 ASAS20 domains [PatientGlobal Assessment, Total Back Pain, Bath AS Functional Index, Bath ASDisease Activity Index questions 5 and 6]), and Major Clinical Response.ASAS40 and ASAS 5/6 outcomes have both been considered as candidatemeasures for determining Major Clinical Response. ASAS40 criteriarepresent a 40% improvement in 5 of 6 domains, without a 20% worseningin the sixth domain. ASAS 5/6 criteria represent a 20% improvement in 5of 6 domains, without a 20% worsening in the sixth domain. Domainsinclude: pain (TBP), function (BASFI), patient's Global Assessment (PGA)of disease activity, inflammation (BASDAI questions 5 & 6), spinalmobility (Bath AS Metrology Index [BASMI]), and C-reactive protein(CRP).

Results

A total of 315 subjects (adalimumab, n=208; placebo, n=107) wereenrolled. Baseline characteristics were similar between subjects in eachtreatment arm. Baseline demographics are shown in Table 12, and thedisposition of subjects enrolled in the study is shown in Table 13.

TABLE 12 Baseline Demographics Adalimumab Placebo 40 mg eow (N = 107) (N= 208) n (%) n (%) Mean Age (years) 43.4 41.7 Sex (males) 79 (73.8) 157(75.5) Race (Whites) 99 (92.5) 202 (97.1) Mean Weight (kg) 79.7 81.9HLA-B27 (positive) 85 (79.4) 163 (78.4) Mean Duration of AS (years) 10.011.3

TABLE 13 Disposition of Subjects Adalimumab Placebo 40 mg eow (%) N (%)Subjects Randomized 107 208 Subjects Treated 107 208 Subjects CompletingWeek 12 103 (96.3)  204 (98.1)  Subjects Completing Week 24 101 (94.4) 195 (93.8)  Prematurely Terminated 4 (3.7) 4 (1.9) at Week 12 AE 2 (1.9)2 (1.0) Withdrew Consent  0 2 (1.0) Lost to Follow Up 1 (0.9)  0 Other 2(1.9) 2 (1.0) Prematurely Terminated at 6 (5.6) 13 (6.3)  Week 24 AE 2(1.9) 5 (2.4) Withdrew Consent 1 (0.9) 5 (2.4) Lost to Follow Up 1 (0.9)2 (1.0) Other 4 (3.7) 4 (1.9)

The number of subjects who met the ASAS partial remission criteria wassignificantly (p≦0.001) higher for the adalimumab group vs. placebogroup at Week 12 (20.7% vs. 3.7%) and at Week 24 (22.1% vs. 5.6%).Adalimumab vs. placebo subjects who met the ASAS5/6 criteria were 48.6%vs. 13.1% (p≦0.001) at Week 12, and 44.7% vs. 12.1% (p≦0.001) at Week24, respectively. The percentage of ASAS40 responders in adalimumabpatients was statistically significantly higher compared to placeboresponders at Week 12 (40.9% vs. 14.0%) and at Week 24 (39.4% vs.14.0%), respectively (p≦0.001, difference between therapies from ANCOVAcalculated using Pearson's Chi-square test). The onset of improvementwas rapid and sustained, as shown in FIG. 8 (ASAS40 response), as animprovement was seen within 2 weeks of administration of adalimumab.

Adverse events (AE), serious AEs, and severe AEs were comparable betweenboth groups, as shown in Table 14. One serious infectious AE was presentin the placebo group. The adalimumab group had no incidence of death,malignant neoplasm, or serious infectious AE.

TABLE 14 Treatment-emergent Adverse Events (AEs) Through Week 24^(†)Adalimumab Placebo 40 mg eow (N = 107) (N = 208) Patients with: n (%) n(%) Any AE 66 (61.7)  163 (78.4)** Serious AE 3 (2.8) 6 (2.9) Severe AE4 (3.7) 6 (2.9) AE leading to discontinuation 2 (1.9) 4 (1.9) of studydrug AE at least possibly drug- 18 (16.8)   74 (35.6)*** relatedInfectious AE 24 (22.4) 70 (33.7) Serious infectious AE 1 (0.9) 0 (0.0)Drug hypersensitivity reaction 1 (0.9) 1 (0.4) ^(†)During administrationof blinded study medication. **, ***Statistically significant at the p =0.01 and p = 0.001 levels, respectively.

Conclusions

Treatment with adalimumab was able to induce both a major clinicalresponse and partial remission over a 24-week period in subjects withAS. Adalimumab was generally safe and well-tolerated. Furthermore,response rates to adalimumab were rapid, as ASAS40 response ratesimproved within the first 2 weeks of treatment.

Example 4 Efficacy of Adalimumab in Active Ankylosing Spondylitis(AS)—Results of the Canadian AS Study

Ankylosing spondylitis (AS) is a common inflammatory rheumatic diseasethat produces progressive spinal stiffness and restriction of mobility.Tumor necrosis factor (TNF) is thought to play a major role in thepathogenesis of AS. No established disease-modifying antirheumatic drug(DMARD) is presently available for the treatment of AS. The purpose ofthe study described herein was to evaluate the efficacy and safety ofadalimumab vs. placebo in patients with active AS.

A 2-year, randomized, placebo-controlled, double-blind, Phase III trialwas conducted at 11 sites in Canada. Patients with active AS who had aninadequate response to at least one NSAID or DMARD were eligible toenroll. The study design is outlined in FIG. 3. Patients were randomizedto receive either placebo or adalimumab 40 mg subcutaneously (sc) everyother week (eow) during an initial 24-week, double-blind period. Patientinclusion criteria were as follows: patients were ≧18 years old,patients had inadequate response to at least one NSAID, and patients hadactive AS, defined by fulfillment of at least 2 of the following 3criteria: BASDAI (Bath Ankylosing Spondylitis Disease Activity Index)score ≧4, Visual Analog Scale (VAS) score for Total Back Pain ≧4,morning stiffness ≧1 hour. Patient exclusion criteria included thefollowing: previously received anti-TNF treatment, radiological evidenceof total spinal ankylosis (bamboo spine), use of previous DMARD within 4weeks of Baseline (other than methotrexate, sulfasalazine, orhydroxychloroquine), intra-articular joint injection withcorticosteroids within 4 weeks of Baseline, and use of other biologicsor investigational therapy within 6 weeks of Baseline. Patients notachieving an ASAS20 (ASsessment in Ankylosing Spondylitis 20) response(calculated by the site) after 12 weeks were eligible, per the protocol,for early escape therapy (EET) of open-label 40 mg adalimumab eow. Anypatient receiving EET was treated as a nonresponder at all subsequentvisits in the statistical analysis.

Primary endpoints of the study were as follows: ASAS20 at Week 12, ASASInternational Working Group Criteria, pain (Total Back Pain [TBP]),function (Bath AS Functional Index [BASFI]), Patient's Global Assessment(PGA) of disease activity, and inflammation (BASDAI questions 5 & 6).Secondary endpoints were ASAS20 at Week 24, BASDAI 20/50/70 at 12 and 24weeks, ASAS50 and ASAS70 responses at 12 and 24 weeks, and PGA ofdisease activity at 12 and 24 weeks.

Results

A total of 82 patients (44 placebo, 38 adalimumab) were enrolled. 80(98%) patients completed the 24-week period. The 2 patients who did notcomplete the 24-week period were from the placebo group. The 2withdrawals from the 24 week period were not related to adverse events.At Week 12, 28 patients randomized to placebo and 20 patients randomizedto adalimumab entered the EET group (64% vs. 53%, respectively). At Week20, 36 patients randomized to placebo and 23 patients randomized toadalimumab entered the EET group (82% vs. 61%, respectively). Baselinecharacteristics were similar between treatment groups, as shown in Table15.

TABLE 15 Baseline Demographics Adalimumab Placebo 40 mg eow (N = 44) (N= 38) Mean age (years) 40.0 41.9 Race (% Caucasian) 42 (95.5) 37 (97.4)Sex (% male) 36 (81.8) 29 (76.3) Mean weight (kg) 78.2 76.1 MeanDuration of AS (years) 12.1 14.5The number of DMARDs at Baseline was likewise similar among treatmentgroups, as shown in Table 16.

TABLE 16 DMARDs at Baseline Adalimumab Placebo 40 mg eow (N = 44) (N =38) Baseline DMARD use^(†)  9 (20.5) 6 (15.8) Methotrexate^(†) 4 (9.1) 4(10.5) Dose (mg/week)* 18.8 ± 894 2000 ± 0 Sulfasalazine^(†)  5 (11.4) 3(7.9)  Dose (mg/day)* 2400 ± 894  2000 ± 0 Leflunomide^(†) 0 0Hydroxychloroquine^(†) 3 (6.8) 0

The ASAS20 response was higher in adalimumab (47%) vs. placebo (27%) atWeek 12. The number of patients achieving ASAS50 and ASAS70 responses atWeek 12 was statistically significantly higher for adalimumab patientscompared to placebo patients (data is shown in Table 17).

TABLE 17 ASAS20/50/70 Scores at Week 12† % of Patients ASAS20 ASAS50ASAS70 Placebo 27.3 6.8 2.3 Adalimumab 47.4 39.5*** 21.1* †Imputed. *,***Statistically significant at p = 0.05 and 0.001 levels, respectively.

Additionally, the ASAS20 response for adalimumab vs. placebo was rapidand was statistically significant (p≦0.01) at Weeks 2, 4, 8, 16, and 20.Adalimumab patients showed significantly greater improvement in TBP,BASFI, PGA and Inflammation scores at Week 12 than did placebo patients(data is shown in Table 18).

TABLE 18 TBP, BASFI, PGA, and Inflammation Scores at Week 12† % Changefrom Baseline TBP BASFI PGA Inflammation Placebo −8.1 0.5 −5.0 −6.0Adalimumab −40.5** −29.6** −36.1** −39.1* †Imputed. *, ***Statisticallysignificant at p = 0.05 and 0.01 levels, respectively.

At Week 12, EET was chosen by 64% of placebo and 53% of adalimumabpatients, and by 82% and 61% of patients, respectively, at Week 20. Theobserved ASAS20 responses at Week 24 in patients initially randomized toadalimumab and placebo were 60% and 73%, respectively. Response to EETwas rapid and sustained in the placebo group. The ASAS20 response forthose patients who switched to EET at Week 12 is shown in FIG. 9. Fiveadalimumab patients with an ASAS20 response at Week 8 had been assessedas nonresponders at Week 12 and were switched to open-label therapy.Four of these 5 had regained an ASAS20 response at Week 16 andmaintained it through Week 24. The observed ASAS20 response time courseis shown in FIG. 10.

Both groups had comparable incidence of adverse events (AEs), with noneof these leading to discontinuation of the study drug. Adalimumabpatients had more infectious AEs (14 [37%] vs. 8 [18%] placebo patients;mostly upper respiratory infections). There was no significantdifference in serious adverse events (SAEs) between groups and no deathsoccurred. Treatment-emergent adverse events (AEs) through Week 24 of thestudy are shown in Table 19, and adverse events with ≧5% incidencethrough week 24 are shown in Table 20.

TABLE 19 Treatment-Emergent Adverse Events (AEs) Through Week 24Adalimumab Placebo 40 mg eow (N = 44) (N = 38) n (%) n (%) Any AE 30(68.2) 33 (86.8) Serious AE 0 (0.0) 1 (2.6) Severe AE 3 (6.8)  4 (10.5)AE leading to discontinuation 0 (0.0) 0 (0.0) of study drug AE at leastpossibly drug-related 13 (29.5) 12 (31.6) Infectious AE  8 (18.2) 14(36.8) Serious infectious AE 0 (0.0) 1 (2.6)

TABLE 20 Adverse Events ≧5% Incidence Through Week 24 Adalimumab Placebo40 mg eow (N = 44) (N = 38) n (%) n (%) Nasopharyngitis  5 (11.4) 7(18.4) Headache 3 (6.8) 5 (13.2) Upper respiratory tract infection 1(2.3) 5 (13.2) Arthralgia  5 (11.4) 4 (10.5) Injection site reaction 4(9.1) 3 (7.9)  Dizziness 2 (4.5) 2 (5.3) 

Conclusions

Adalimumab was efficacious in reducing the signs and symptoms of activeAS and was generally well-tolerated. The possibility of early escapetherapy influenced the results beyond Week 8.

Example 5 Adalimumab Improves Health-Related Quality of Life in Patientswith Active Ankylosing Spondylitis—Study H

Ankylosing Spondylitis (AS) is a common inflammatory disease thatproduces spinal stiffness and restriction of mobility. The clinicalsymptoms and subsequent disease progression of AS may result infunctional limitations and impairment in HRQL. Tumor necrosis factor(TNF) has been reported to play a major role in the pathogenesis of AS.Adalimumab is a fully human monoclonal antibody targeting TNF, currentlyapproved for the treatment of rheumatoid arthritis and psoriaticarthritis in the US and Europe. Adalimumab is currently pending approvalfrom the FDA and EMEA for AS. Study H was a phase III, randomized,placebo-controlled, double-blind, multi-center study designed to assessthe efficacy and safety of adalimumab in the treatment of active AS insubjects who had an inadequate response, or were intolerant to,treatment with at least one nonsteroidal anti-inflammatory drug (NSAID);patients may have had an inadequate response to at least one DMARD(disease modifying anti-rheumatic drug). The objective of the studydescribed herein was to assess the effect of adalimumab in improvingfunction and HRQOL in patients with active AS who were treated withadalimumab in Study H.

Subjects were randomized to either adalimumab 40 mg every other week orplacebo for 24 weeks (study design for Study H is outlined in FIG. 7).An early escape option to open label 40 mg adalimumab sc eow wasavailable at Week 12, or 16, or 20. Disease activity was evaluated usingthe Bath Ankylosing Spondylitis Disease Activity Index (BASDAI)patient-reported questionnaire. Assessment in AS (ASAS) 20 criteria wasthe primary efficacy measure. Functioning and HRQL were assessed bypatient-reported questionnaires, including Bath AS Functional Index(BASFI), SF-36 (Short Form-36) and Ankylosing Spondylitis Quality ofLife Questionnaire (ASQoL). Patient-reported questionnaires utilized inthis study are described in greater detail below.

The BASDAI is a six-item measure of disease activity and includesquestions on fatigue, spinal pain, peripheral arthritis, enthesitis, andduration and severity of morning stiffness. It is a well-establishedinstrument widely used in clinical studies to evaluate AS diseaseactivity. Items are related to the past 7 days and are answered on a 10cm visual analogue scale (VAS), with score ranges from 0 (none) to 10(very severe). Instrument was administered at Baseline, and at Weeks 2,4, 8, 12, 16, 20 and 24.

The BASFI is a set of 10 questions designed to determine the degree offunctional limitation in those with AS. Items are related to the past 7days and are answered on a 10 cm visual analogue scale (VAS). The scoreranges from 0 to 100, with lower scores reflecting less functionlimitation. Instrument was administered at Baseline, and at Weeks 2, 4,8, 12, 16, 20 and 24.

SF-36 is a widely applied instrument for measuring health status andconsists of 8 domains: physical function, bodily pain, rolelimitations-physical, general health, vitality, social function, rolelimitations-emotional, and mental health. The Recall period is 4 weeks,and domain scores range from 0-100, with higher scores reflecting betterhealth status. A 5-10 point change in domain scores is consideredclinically meaningful (Kosinski M et al. Arth Rheum 2000; 7:1478-1487).SF-36 also contains 2 summary scores, the mental component summary (MCS)and the physical component summary (PCS); a 2.5-5 point change insummary scores is considered clinically meaningful (Kosinski M et al.Arth Rheum 2000; 7:1478-1487). This instrument was administered atBaseline, and at Weeks 12 and 24.

The ASQoL is a disease-specific instrument designed to measure HRQL insubjects with AS, developed on a needs-based model. Subjects are askedto answer 18 yes/no items concerning the impact of AS “at this moment”.The ASQoL has a total score ranging from 0 to 18, with lower scoresrepresenting better AS-specific quality of life. The instrument has goodreliability and construct validity across several different ASpopulations (Doward L C, et al. Ann Rheum Dis 2003; 62:20-26; Haywood KL, et al J Rheumatol 2003; 30:764-773; van Tubergen A et al. ArthritisRheum 2002; 47:8-16; Marzo-Ortega H, et al. Arthritis Rheum 2001;44(9):2112-2117). The pre-specified minimum important difference (MID)has been suggested to be a change of 1-2 points, 10% of the total score(Haywood K L, et al J Rheumatol 2003; 30:764-773; Haywood K L, et al.Rheum 2002; 41:1295-1302). Assessments were made at Baseline and atWeeks 2, 12, and 24.

Results

A total of 315 subjects (adalimumab, N=208; placebo, N=107) wereenrolled. The disposition of subjects and study completion rates areshown above in Table 13. At baseline, adalimumab and placebo arms hadcomparable demographic and disease characteristics, SF-36 (PCS, MCS),and ASQoL scores, as shown in Table 12 above. Functioning and HRQOLassessment at baseline is shown in Table 21. Baseline SF-36 PCS scores(placebo 31.7, adalimumab 33.1) were almost 20 points lower than theU.S. general population norm (50.0), indicating a substantial impairmentof physical health status, while baseline mental health status measuredby SF-36 MCS scores (placebo 44.7, adalimumab 43.5) were close to thepopulation norm.

TABLE 21 Functioning and HRQoL Assessment at Baseline Adalimumab Placebo40 mg eow (N = 107) (N = 208) p-value^(†) SF-36 PCS 31.8 32.9 0.519ASQoL 10.6 10.2 0.343 SF-36 MCS 44.4 43.4 0.407 SF-36 Subscales 0.495Physical Function 45.5 47.9 0.524 Role-Physical 19.9 20.5 0.214 SocialFunction 53.4 57.2 0.323 General Health 41.0 43.4 0.491 Bodily Pain 29.831.7 0.544 Vitality 34.0 32.6 0.491 Role-Emotional 56.8 53.2 0.701Mental Health 62.5 61.3 0.567 BSDAI Fatigue 6.7 6.5 0.846 *Unadjustedmeans ^(†)Differences between treatment groups were assessed usinganalysis of variance with treatment group and baseline scores ascovariates

At week 12 and 24, adalimumab treatment demonstrated statisticallysignificant ASAS20 response (the primary efficacy measurement) comparedwith placebo, and the onset of improvement by adalimumab was rapid andsustained (data is shown in FIG. 11). At Week 12, adalimumab patientsshowed statistically significantly greater improvement in SF-36 PCSscores than did placebo patients. These statistically significantimprovements increased through Week 24. Change in SF-36 PCS scores foradalimumab patients well exceeded the MID value and suggested asustained clinically important improvement; placebo groups did notachieve the MID for SF-36 PCS scores. The proportion of patients whoachieved the MID in SF-36 PCS scores was statistically significant inadalimumab vs. placebo at 12 weeks (65.0 vs. 37.6, respectively,p≦0.001) and 24 weeks (67.3 vs. 39.6, respectively, p≦0.001) (data isshown in Table 22). Neither the adalimumab group nor the placebo groupexperienced any significant change in MCS scores at Week 12 or Week 24.The proportion of patients that achieved the MID in change of MCS scoreswas similar in both the adalimumab and placebo groups.

TABLE 22 Change from Baseline in SF-36 PCS Scores at 12 Weeks and 24Weeks Mean Change from Baseline^(†) Placebo Adalimumab Week 12 1.66.9*** Week 24 1.9 7.4*** ^(†)LOCF Minimum Important Difference = 3(Kosinski M et al. Arth Rheum 2000; 7: 1478-1487). ***Statisticallysignificant at p = 0.001 level. p-value for difference between therapiesfrom ANCOVA.

At both Weeks 12 and 24, patients treated with adalimumab showedstatistically significant improvement in 7 out of the 8 subscalescompared with those treated with placebo, including the 4 subscales thatare most closely related to the SF-36 PCS (Physical Functioning,Role-Physical, Bodily Pain, and General Health) and 3 of the 4 subscalesthat are most closely related to the SF-36 MCS (Vitality, SocialFunctioning, and Role-Emotional) (12-Week data is shown in Table 23,24-Week data is shown in Table 24). The differences at Weeks 12 and 24are also considered to be clinically meaningful, based on the MID of5-10 point change scores.

TABLE 23 Change from Baseline in SF-36 Domain Scores at 12 Weeks^(†)Mean Change Placebo Adalimumab Physical Functioning 3 11.8***Role-Physical 7.9 25.3*** Social Functioning 6.3 8.7*  General Health1.4 8***  Bodily Pain 6.7 19.2*** Vitality 6.4 13.1**  Role-Emotional3.2 14.7*  Mental Health 4.3 4.6  ^(†)LOCF unadjusted means Differencesbetween treatment groups were assessed using an analysis of covariancewith treatment group and baseline scores as covariates. *, **, and ***are statistically significant at the p < 0.05, p < 0.01, and p < 0.001levels, respectively.

TABLE 24 Change from Baseline in SF-36 Domain Scores at 24 Weeks^(†)Mean Change Placebo Adalimumab Physical Functioning 4.2 13.2***Role-Physical 9.2 27.6*** Social Functioning 6.5 12.2*** General Health1.2 8.8*** Bodily Pain 7.1 20.7*** Vitality 5.6 14.7*** Role-Emotional3.5 15.9* Mental Health 4.7 5.9 ^(†)LOCF unadjusted means Differencesbetween treatment groups were assessed using an analysis of covariancewith treatment group and baseline scores as covariates. *, **, and ***are statistically significant at the p < 0.05, p < 0.01, and p < 0.001levels, respectively.

More adalimumab patients experienced a statistically significantimprovement in BASFI compared with placebo patients at Week 12 and Week24 (data is shown in Table 25).

TABLE 25 Change from Baseline in BASFI Scores at 12 Weeks and 24Weeks^(†) Mean Change at Baseline‡ Placebo Adalimumab Week 12 −5.05−17.46*** Week 24 −5.16 −18.7*** ^(†)LOCF ‡unadjusted means***Statistically significant at the pp < 0.001, placebo vs. adalimumab.p-value for differences between therapies from an ANCOVA with therapyand baseline values as a covariate.

At Week 12 and Week 24, adalimumab patients showed greater improvementin overall HRQL as measured by ASQoL scores when compared to placebopatients (data is shown in Table 26). The change in ASQoL scoresexceeded the prior MID value and suggested a sustained clinicallyimportant improvement. The proportion of adalimumab patients thatachieved the MID in ASQoL score was statistically significantly higherthan the proportion of placebo patients. The proportion of patients whoachieved the MID in ASQoL scores was statistically significant inadalimumab vs. placebo at 12 weeks (59.1 vs. 42.1, respectively, p<0.01)and 24 weeks (65.4 vs. 42.1, respectively, p<0.001).

TABLE 26 Change from Baseline in ASQoL Scores at 12 Weeks and 24Weeks^(†) Mean Change at Baseline‡ Placebo Adalimumab Week 12 −1.0−3.1*** Week 24 −1.1 −3.6*** ^(†)LOCF ‡unadjusted means MinimumImportant Difference = −2 (Haywood K. L. et al. J. Rheumatol. 2003; 30:764-773; Haywood K. L. et al. Rheum. 2002; 41: 1295-1302).***Statistically significant at the p = 0.001 level, p-value assessedusing analysis of variance within treatment group and baseline scores ascovariates.

Conclusions

These results suggest that adalimumab therapy may improve physicalhealth status and overall HRQL in AS patients. After 12 and 24 weeks ofadalimumab therapy, patients reported statistically significant andclinically meaningful improvements in physical functioning as measuredby SF-36 PCS and BASFI compared with placebo. At 12 and 24 weeks,patients treated with adalimumab reported statistically significant andclinically meaningful improvements in overall HRQL as measured by ASQoLcompared with those treated with placebo.

Example 6 Adalimumab Therapy Results in Significant Reduction of Signsand Symptoms in Subjects with Ankylosing Spondylitis

The aim of the following study (Study H) was to assess the efficacy andsafety of adalimumab in the treatment of AS. The study design of Study His outlined in FIG. 7, and included a double-blind placebo-controlled 24week study followed by a continuous open label study (shown in FIG. 7).Details regarding Study H are also provided in the above examplesreferencing this study, as well as below.

Inclusion criteria were the following: ≧18 years of age; AS based onmodified New York criteria; inadequate response to ≧1 NSAID; and activeAS, as diagnosed by 2 of the 3 following symptoms: Bath AS DiseaseActivity Index (BASDAI) score ≧4; Visual Analog Scale (VAS) score forTotal Back Pain (TBP)≧4; and Morning stiffness ≧1 hour. Patients notachieving an ASAS 20 response after 12 weeks were eligible for earlyescape therapy (EET) of open-label 40 mg adalimumab eow. Any patientreceiving EET was treated as a nonresponder at all subsequent visits inthe statistical analysis.

The primary endpoint for monitoring reduction of signs and symptoms wasASAS 20 at Week 12. Major secondary endpoints were ASAS 40, ASAS 5/6,and ASAS Partial Remission Criteria. Outcome measures were assessed atWeeks 12 and 24.

ASAS20 improvement criteria included assessment of patient global, pain,function, and inflammation, and required an improvement of ≧20% and ≧1unit in at least 3 of these domains, without a worsening of ≧20% and ≧1unit in the remaining domain (see Anderson et al. (2001) Arthritis Rheum44:1876-1886).

ASAS 40 improvement criteria included an assessment of the same fourdomains as for ASAS20, and required an improvement of ≧40% and ≧2 unitsin at least 3 domains, with no worsening at all in the remaining domain(see Brandt et al. (2004) Ann Rheum 63:1438-1444).

ASAS 5/6 improvement criteria included an improvement of ≧20% in atleast 5 of the following 6 domains: patient global, pain, function,inflammation, CRP, and spinal mobility (see Brandt et al. (2004) AnnRheum 63:1438-1444).

ASAS partial remission criteria include a value of <2 units in all fourof the following domains: patient global, pain, function, andinflammation (see Anderson et al. (2001) Arthritis Rheum 44:1876-1886).

Baseline demographics are shown in Table 12 above, and concomitantdiseases or symptoms present at baseline are shown in Table 27.

TABLE 27 Concomitant Diseases or Symptoms at Baseline Adalimumab Placebo40 mg eow (N = 107) (N = 208) n (%) n (%) History of inflammatory bowel6 (5.6) 21 (10.1) disease† Peripheral arthritis‡ 44 (44.1) 75 (36.1)History of psoriasis† 17 (15.9) 16 (7.7)  History of uveitis† 27 (25.2)68 (32.7) *No significant differences between groups except forpsoriasis ^(†)Stable for at least four weeks prior to Baseline ^(‡)Atleast one SJC at Baseline

Baseline disease activity is shown in Table 28. Concomitant Treatment atbaseline is shown in Table 29. The disposition of subjects in the trialis shown above in Table 13. Patients receiving early escape open-labeltherapy are shown in Table 30.

TABLE 28 Baseline Disease Activity Adalimumab Placebo 40 mg eow (N =107) (N = 208) BASDAI score* 6.3 6.3 BASDAI categories^(†) <4 12 (11.2) 25 (12.0) 4-6 30 (28.0)  62 (29.8) >6 65 (60.7) 121 (58.2) Total BackPain VAS 6.7 6.4 Morning Stiffness 6.7 6.7 BASFI score* 5.6 5.2 CRP(mg/dL)* 2.2 1.8 Patients with elevated CRP^(†‡) 75 (70.1) 138 (66.3)*Mean ^(†)n (%) ^(‡)Normal CRP range≦0.494 mg/dl

TABLE 29 Concomitant Treatment at Baseline Adalimumab Placebo 40 mg eow(N = 107) (N = 208) Baseline DMARD use* 22 (20.6) 40 (19.2) Methotrexate8 (7.5) 20 (9.6)  Sulfasalazine 15 (14.0) 26 (12.5) Leflunomide 1 (0.9)0 Oral corticosteroids 6 (5.6) 25 (12.0) NSAIDs 84 (78.5) 166 (79.8)  *n(%)

TABLE 30 Patients Receiving Early Escape Open-label Therapy AdalimumabPlacebo 40 mg eow (N = 107) (N = 208) Visit n (%) n (%) Total 74 (69.2)81 (38.9) Week 12 55 (51.4) 54 (26.0) Week 14 11 (10.3) 5 (2.4) Week 166 (5.6) 10 (4.8)  Week 18 0 (0.0) 1 (0.5) Week 20 2 (1.9) 8 (3.8) Week22 0 (0.0) 3 (1.4)

A statistically significant change from baseline was observed betweenthe adalimumab and placebo patient treatment groups in the followingASAS20 components: patient's global assessment of disease activity,total back pain, and inflammation. The baseline mean score for patient'sglobal assessment of disease activity was 64.5 for the placebo treatmentgroup and 63.2 for the adalimumab treatment group. The percentage changefrom baseline observed in the placebo group was 6.5 at Week 12, and 8.7at Week 24 (N=107). The percentage change from baseline observed in theadalimumab group was −39.1 at Week 12, and −37.8 at Week 24 (N=208;difference between placebo vs. adalimumab is significant at p=0.001level, determined by ANCOVA).

The baseline mean score for total back pain was 67.2 for the placebotreatment group and 64.6 for the adalimumab treatment group. Thepercentage change from baseline observed in the placebo group was −9.5at Week 12 and −10.0 at Week 24 (N=107). The percentage change frombaseline observed in the adalimumab group was −40.5 at Week 12, and−42.4 at Week 24 (N=208; difference between placebo vs. adalimumab issignificant at p=0.001 level, determined by ANCOVA).

The baseline mean score for inflammation (the mean of BASDAI questions 5and 6) was 6.7 for both the placebo and adalimumab treatment groups. Thepercentage change from baseline observed in the placebo group was −15.2at Week 12 and −12.5 at Week 24 (N=107). The percentage change frombaseline observed in the adalimumab group was −41.7 at Week 12, and−42.9 at Week 24 (N=208; difference between placebo vs. adalimumab issignificant at p=0.001 level, determined by ANCOVA).

The mean change in ASAS 20, ASAS 40, and ASAS 5/6 from baseline at Week12 and Week 24 is shown in Table 31. BASDAI 50 at Week 12 and Week 24 isshown in Table 32.

TABLE 31 ASAS20†, ASAS40‡, and ASAS 5/6‡ Mean Change from BaselineASAS20 ASAS40 ASAS5/6 Week 12 Placebo 20.6 14.0 13.1 Adalimumab 58.2***40.9*** 48.6*** Week 24 Placebo 18.7 14.0 12.1 Adalimumab 50.5***39.4*** 44.7*** †Imputed; ‡LOCF ***Statistically significant at p <0.001 level (ANCOVA)

TABLE 32 BASDAI 50 % of Patients Placebo Adalimumab (N = 107) (N = 208)Week 12 15.9 45.2*** Week 24 15.0 42.3*** ***Statistically significantat p = 0.001 level (Pearson's Chi-Square test). Patients with missingdata at Weeks 12 and 24 are counted as non-responders.

The percentage of patients achieving partial remission at Week 12 andWeek 24 is shown in Table 33. The mean change in BASDAI, BASFI, andBASAMI from baseline at Week 24 is shown in Table 34.

TABLE 33 Partial Remission‡ Partial Remission % of Patients† PlaceboAdalimumab (N = 107) (N = 208) Week 12 3.7 20.7*** Week 24 5.6 22.1***†LOCF; ‡Partial remission is defined as a value <20 on a 0-100 scale ineach of the four ASAS domains. ***Statistically significant at p = 0.001level (Pearson's Chi-Square test).

TABLE 34 BASDAI, BASFI, and BASMI at Week 24 Mean change from BaselinePlacebo Adalimumab BASDAI −0.8 −2.6*** BASFI −0.5 −1.87*** BASMI 0.0−0.6 †LOCF ***Statistically significant at p < 0.001 level (ANCOVA)

A Subgroup Analysis of ASAS 20 responders with Total Spinal Ankylosis isshown in Table 35.

TABLE 35 Subgroup Analysis-Total Spinal Ankylosis ASAS20 Responders (%of Patients) Yes No Week 12 Placebo 0 (n = 5) 21.6 (n = 102) Adalimumab50 (n = 6) 58.2 (n = 201) Week 24 Placebo 0 (n = 5) 19.6 (n = 102)Adalimumab 66.7 (n = 6) 49.8 (n = 201)

The Maastricht AS Enthesitis Score (MASES), which assesses the patientsresponse to firm palpation at 13 points in the chest, hip, and footregions, was measured at Weeks 12 and 24. Possible total scores rangefrom 0-13, with a score of 0 indicating no pain, and a score of 1indicating pain. Consequently, a decrease in MASES is representative ofimprovement. The mean baseline MASES was 6.7 in the placebo treatmentgroup, and 6.3 in the adalimumab treatment group. The mean change frombaseline MASES (LOCF) in the placebo group was −1.3 at Week 12 and −1.6at Week 24 (N=106). The mean change from baseline MASES in theadalimumab group was −2.7 at Week 12 (N=204; statistically significantat p<0.05 vs. placebo as determined by ANCOVA), and −3.2 at Week 24(N=205; statistically significant at p<0.01 vs. placebo as determined byANCOVA).

Adverse events with a ≧5% incidence through Week 24 are shown in Table36. Treatment-emergent adverse events through week 24 are shown in Table37.

TABLE 36 Adverse Events ≧5% Incidence Through Week 24^(†) AdalimumabPlacebo 40 mg eow (N = 107) (N = 208) MedDRA preferred term: n (%) n (%)P-value‡ Nasopharyngitis 8 (7.5) 26 (12.5) 0.249 Injection site reaction3 (2.8) 22 (10.6) 0.015* Headache 9 (8.4) 20 (9.6)  0.838 ^(†)Duringadministration of blinded study medication ^(‡)Fischer's exact test (2tail) *Statistically significant at p ≦ 0.05 level

TABLE 37 Treatment-emergent Adverse Events (AEs) Through Week 24^(†)Adalimumab Placebo 40 mg eow (N = 107) (N = 208) Patients with: n (%) n(%) p-value^(‡) Any AE 66 (61.7) 163 (78.4)  0.002** Serious AE 3 (2.8)6 (2.9) NS Severe AE 4 (3.7) 6 (2.9) NS AE leading to discontinuation 2(1.9) 4 (1.9) NS of study drug AE at least possibly 18 (16.8) 74 (35.6)<0.001*** drug-related Infections AE 24 (22.4) 70 (33.7) NS Seriousinfectious AE 1 (0.9) 0 (0.0) NS Drug hypersensitivity reaction 1 (0.9)1 (0.4) NS Malignant neoplasm 0 (0.0) 0 (0.0) NS Death 0 (0.0) 0 (0.0)NS ^(†)During administration of blinded study medication ^(‡)Onlystatistically significant p-values are shown **, ***Statisticallysignificant at the p = 0.01 and p = 0.001 levels, respectively(Pearson's Chi-Square test)

Laboratory tests were performed through Week 24. There were smallchanges in hemoglobin, platelets, neutrophils and lymphocyte counts inadalimumab-treated patients. Adalimumab patients had significantlyhigher levels of liver enzymes (ALT, AST) and total bilirubin comparedwith placebo; however, these changes were small. Baseline and maximumALT values through Week 24 are shown in Table 38. Adalimumab patientsalso had a significantly greater decrease in C-reactive protein (CRP) atWeeks 12 and 24. The baseline mean CRP was 2.16 mg/dL in the placebotreatment group and 1.76 mg/dL in the adalimumab treatment group. Themean change from baseline (LOCF) in CRP (mg/dL) among patients in theplacebo group was −0.08 at Week 12 and −0.06 at Week 24 (N=105). Themean change from baseline among patients in the adalimumab group was−1.28 at Week 12 and −1.25 at Week 24 (N=204; statistically significantvs. placebo at p≦0.001 level as determined by ANCOVA).

TABLE 38 Baseline and Maximum ALT Values Through Week 24 Maximum ALTValue <1.5 × ≧1.5 × ULN-<3.0 × ≧3.0 × ULN-<8.0 × ≧8.0 × ULN ULN ULN ULNBaseline ALT Value n (%) n (%) n (%) n (%) Placebo (N = 107) <1.5 × ULN104 (97.2) 2 (1.9) 0 0 ≧1.5 × ULN-<3.0 × ULN 0 0 0 0 ≧3.0 × ULN-<8.0 ×ULN 0 1 (0.9) 0 0 ≧8.0 × ULN 0 0 0 0 Adalimumab 40 mg eow (N = 208) <1.5× ULN 175 (84.1) 23 (11.1) 2 (1.0) 1 (0.5) ≧1.5 × ULN-<3.0 × ULN  1(0.5) 1 (0.5) 3 (1.4) 0 ≧3.0 × ULN-<8.0 × ULN 0 2 (1.0) 0 0 ≧8.0 × ULN 00 0 0

Conclusions

Adalimumab was generally well tolerated in patients with active AS. Thesafety profile of adalimumab in the Study H Trial was consistent withthat observed in RA and PsA trials. Adalimumab was effective in treatingsubjects with active AS. Adalimumab showed similar efficacy in patientswith and without Total Spinal Ankylosis (TSA).

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims. The contents of allreferences, patents and published patent applications cited throughoutthis application are incorporated herein by reference.

1. A method of decreasing pain and fatigue in a subject having AScomprising administering a human TNFα antibody, or antigen-bindingportion thereof, to the subject such that pain and fatigue aredecreased.
 2. The method of claim 1, wherein the decrease in fatigue inthe subject is determined by a score selected from the group consistingof FACIT-F, BASDAI, and SF-36.
 3. The method of claim 1, wherein thedecrease in fatigue is determined by a decrease of at least about 1.9 ina BASDAI score of the subject or a decrease of at least about 2.0 in aBASDAI score of the subject.
 4. (canceled)
 5. A method of determiningthe efficacy of a human TNFα antibody, or antigen-binding fragmentthereof, for treating ankylosing spondylitis (AS) in a subjectcomprising determining a partial remission rate of a patient populationhaving AS and who was administered the human TNFα antibody, orantigen-binding fragment thereof, wherein a partial remission rate of atleast about 20% of the patient population indicates that the human TNFαantibody, or antigen-binding fragment thereof, is an effective humanTNFα antibody, or antigen-binding fragment thereof, for the treatment ofAS.
 6. A method of determining the efficacy of a human TNFα antibody, orantigen-binding fragment thereof, for treating ankylosing spondylitis(AS) in a subject comprising determining a Bath Ankylosing SpondylitisDisease Activity Index (BASDAI) response of a patient population havingAS and who was administered the human TNFα antibody, or antigen-bindingfragment thereof, wherein the BASDAI response selected from the groupcomprising a BASDAI 20 response in at least 60% of the patientpopulation, a BASDAI 20 response in at least about 70% of the patientpopulation, a BASDAI 20 response in at least about 80% of the patientpopulation, a BASDAI 20 response in at least about 85% of the patientpopulation, a BASDAI 50 response of at least about 23% of the patientpopulation, a BASDAI 50 response of at least about 30% of the patientpopulation, a BASDAI 50 response of at least about 40% of the patientpopulation, a BASDAI 50 response of at least about 50% of the patientpopulation, a BASDAI 50 response of at least about 60% of the patientpopulation, a BASDAI 70 response in at least about 10% of the patientpopulation, a BASDAI 70 response in at least about 20% of the patientpopulation, a BASDAI 70 response in at least about 30% of the patientpopulation, a BASDAI 70 response in at least about 40% of the patientpopulation, and a BASDAI 70 response in at least about 45% of thepatient population, indicates that the human TNFα antibody, orantigen-binding fragment thereof, is an effective human TNFα antibody,or antigen-binding fragment thereof, for the treatment of AS. 7-19.(canceled)
 20. A method of determining the efficacy of a human TNFαantibody, or antigen-binding fragment thereof, for treating ankylosingspondylitis (AS) in a subject comprising determining a Assessment inAnkylosing Spondylitis (ASAS) response of a patient population having ASand who was administered the human TNFα antibody, or antigen-bindingfragment thereof, wherein the ASAS response selected from the groupconsisting of an ASAS20 response in at least about 27% of the patientpopulation, an ASAS20 response in at least about 50% of the patientpopulation, an ASAS20 response in at least about 55% of the patientpopulation, an ASAS20 response in at least about 70% of the patientpopulation, an ASAS40 response in at least about 10% of the patientpopulation, an ASAS40 response in at least about 20% of the patientpopulation, an ASAS40 response in at least about 30% of the patientpopulation, an ASAS40 response in at least about 45% of the patientpopulation, an ASAS70 response in at least about 5% of the patientpopulation, an ASAS70 response in at least about 20% of the patientpopulation, an ASAS70 response in at least about 23% of the patientpopulation, an ASAS70 response in at least about 30% of the patientpopulation, and an ASAS70 response in at least about 40% of the patientpopulation, indicates that the human TNFα antibody, or antigen-bindingfragment thereof, is an effective human TNFα antibody, orantigen-binding fragment thereof, for the treatment of AS. 21-33.(canceled)
 34. The method of claim 6 or 20, further comprisingadministering the effective human TNFα antibody, or antigen-bindingfragment thereof, to a subject for the treatment of AS.
 35. A method oftreating AS in a subject comprising administering an effective humanTNFα antibody, or antigen-binding fragment thereof, to the subject suchthat AS is treated, wherein the administration of the effective humanTNFα antibody, or antigen-binding fragment thereof, was previouslyidentified as resulting in a response selected from the group consistingof a BASDAI 20 response in at least about 60% of a patient populationhaving AS, a BASDAI 50 response in at least about 23% of a patientpopulation having AS, a BASDAI 70 response in at least about 10% of apatient population having AS, an ASAS20 response in at least about 50%of a patient population having AS, an ASAS50 response in at least about39% of a patient population having AS, and an ASAS70 response in atleast about 5% of a patient population having AS. 36-41. (canceled) 42.The method of any one of claims 6, 20 or 35, wherein the TNFα antibody,or antigen-binding portion thereof, is a multivalent antibody.
 43. Themethod of any one of claims 6, 20 or 35, wherein the human TNFαantibody, or antigen-binding portion thereof, is golimumab.
 44. Themethod of any one of claims 1, 5, 6, 20, 35 or 42, wherein the humanTNFα antibody, or antigen-binding portion thereof, is selected from thegroup consisting of: i) a human TNFα antibody, or antigen-bindingportion thereof, that dissociates from human TNFα with a K_(d) of 1×10⁻⁸M or less and a K_(off) rate constant of 1×10⁻³ s⁻¹ or less, bothdetermined by surface plasmon resonance, and neutralizes human TNFαcytotoxicity in a standard in vitro L929 assay with an IC₅₀ of 1×10⁻⁷ Mor less; ii) a human TNFα antibody, or antigen-binding portion thereof,that: a) dissociates from human TNFα with a K_(off) rate constant of1×10⁻³ s⁻¹ or less, as determined by surface plasmon resonance; b) has alight chain CDR3 domain comprising the amino acid sequence of SEQ ID NO:3, or modified from SEQ ID NO: 3 by a single alanine substitution atposition 1, 4, 5, 7 or 8 or by one to five conservative amino acidsubstitutions at positions 1, 3, 4, 6, 7, 8 and/or 9; c) has a heavychain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4, ormodified from SEQ ID NO: 4 by a single alanine substitution at position2, 3, 4, 5, 6, 8, 9, 10 or 11 or by one to five conservative amino acidsubstitutions at positions 2, 3, 4, 5, 6, 8, 9, 10, 11 and/or 12; iii) ahuman TNFα antibody, or antigen-binding portion thereof, that comprisesa light chain variable region (LCVR) having a CDR3 domain comprising theamino acid sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by asingle alanine substitution at position 1, 4, 5, 7 or 8, and comprises aheavy chain variable region (HCVR) having a CDR3 domain comprising theamino acid sequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by asingle alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11;iv) a human TNFα antibody, or antigen-binding portion thereof, a lightchain variable region (LCVR) comprising the amino acid sequence of SEQID NO: 1 and a heavy chain variable region (HCVR) comprising the aminoacid sequence of SEQ ID NO: 2; and v) adalimumab. 45-48. (canceled) 49.The method of any one of claims 1, 5, 6, 20 or 35, wherein the humanTNFα antibody, or an antigen-binding portion thereof, is administered tothe subject on a biweekly dosing regimen.
 50. The method of any one ofclaims 1, 5, 6, 20 or 35, wherein the human TNFα antibody, or anantigen-binding portion thereof, is administered in a dose of about 40mg.
 51. The method of any one of claims 1, 5, 6, 20 or 35 wherein thehuman TNFα antibody, or an antigen-binding portion thereof, isadministered to the subject subcutaneously.